Use of Small Molecules to Enhance Mafa Expression in Pancreatic Endocrine Cells

ABSTRACT

The present invention provides methods, cell cultures and differentiation media to promote differentiation of pluripotent stem cells to pancreatic endocrine cells of a mature phenotype. The resulting pancreatic endocrine cells express single hormonal insulin, PDX1, NKX6.1, and MAFA. In one or more differentiation stages, culturing may be carried out in a culture vessel at the air-liquid interface.

CROSS REFERENCE TO RELATED APPLICATION

The present application claims the benefit of U.S. Provisional PatentApplication Ser. No. 61/994,259, filed May 16, 2014, which isincorporated herein by reference in its entirety for all purpose.

FIELD OF THE INVENTION

The present invention relates to methods for, and cells and populationsresulting from, the differentiation of pluripotent stem cells. Inparticular, the invention relates to the use of certain small moleculesto generate pancreatic endocrine cells, and populations of such cells,that exhibit increased expression of MAFA.

BACKGROUND

Advances in cell-replacement therapy for Type I diabetes mellitus and ashortage of transplantable islets of Langerhans have focused interest ondeveloping sources of insulin-producing cells, or beta (β) cells,appropriate for engraftment. One approach is the generation offunctional β cells from pluripotent stem cells, such as, embryonic stemcells.

In vertebrate embryonic development, a pluripotent cell gives rise to agroup of cells comprising three germ layers (ectoderm, mesoderm, andendoderm) in a process known as gastrulation. Tissues such as, thyroid,thymus, pancreas, gut, and liver will develop from the endoderm via anintermediate stage. The intermediate stage in this process is theformation of definitive endoderm.

By the end of gastrulation, the endoderm is partitioned intoanterior-posterior domains that can be recognized by the expression of apanel of factors that uniquely mark anterior, mid, and posterior regionsof the endoderm. For example, HHEX, and SOX2 identify the anteriorregion while CDX1, 2, and 4 identify the posterior region of theendoderm.

Migration of endoderm tissue brings the endoderm into close proximitywith different mesodermal tissues that help in regionalization of thegut tube. This is accomplished by a plethora of secreted factors, suchas fibroblast growth factors (“FGFs”), WNTS, transforming growth factorbetas (“TGF-βs”), retinoic acid, and bone morphogenic protein (“BMP”)ligands and their antagonists. For example, FGF4 and BMP promote CDX2expression in the presumptive hindgut endoderm and repress expression ofthe anterior genes HHEX and SOX2 (2000 Development, 127:1563-1567). WNTsignaling has also been shown to work in parallel to FGF signaling topromote hindgut development and inhibit foregut fate (2007 Development,134:2207-2217). Lastly, secreted retinoic acid by mesenchyme regulatesthe foregut-hindgut boundary (2002 Curr. Biol., 12:1215-1220).

The level of expression of specific transcription factors may be used todesignate the identity of a tissue. During transformation of thedefinitive endoderm into a primitive gut tube, the gut tube becomesregionalized into broad domains that can be observed at the molecularlevel by restricted gene expression patterns. The regionalized pancreasdomain in the gut tube shows a very high expression of PDX and very lowexpression of CDX2 and SOX2. PDX1, NKX6.1/PTF1A, and NKX2.2 are highlyexpressed in pancreatic tissue and expression of CDX2 is high inintestinal tissue.

Formation of the pancreas arises from the differentiation of definitiveendoderm into pancreatic endoderm. Dorsal and ventral pancreatic domainsarise from the foregut epithelium. Foregut also gives rise to theesophagus, trachea, lungs, thyroid, stomach, liver, and bile ductsystem.

Cells of the pancreatic endoderm express the pancreatic-duodenalhomeobox gene PDX1. In the absence of PDX1, the pancreas fails todevelop beyond the formation of ventral and dorsal buds. Thus, PDX1expression marks a critical step in pancreatic organogenesis. The maturepancreas contains both exocrine and endocrine tissues arising from thedifferentiation of pancreatic endoderm.

D'Amour et al. describe the production of enriched cultures of humanembryonic stem cell-derived definitive endoderm in the presence of ahigh concentration of activin and low serum (Nature Biotechnology 2005,23:1534-1541; U.S. Pat. No. 7,704,738). Transplanting these cells underthe kidney capsule of mice resulted in differentiation into more maturecells with characteristics of endodermal tissue (U.S. Pat. No.7,704,738). Human embryonic stem cell-derived definitive endoderm cellscan be further differentiated into PDX1 positive cells after addition ofFGF-10 and retinoic acid (U.S. Patent App. Pub. No. 2005/0266554).Subsequent transplantation of these pancreatic precursor cells in thefat pad of immune deficient mice resulted in the formation of functionalpancreatic endocrine cells following a 3-4 months maturation phase (U.S.Pat. Nos. 7,534,608 and 7,993,920).

Fisk et al. report a system for producing pancreatic islet cells fromhuman embryonic stem cells (U.S. Pat. No. 7,033,831). In this case, thedifferentiation pathway was divided into three stages. Human embryonicstem cells were first differentiated to endoderm using a combination ofsodium butyrate and activin A (U.S. Pat. No. 7,326,572). The cells werethen cultured with BMP antagonists, such as Noggin, in combination withEGF or betacellulin to generate PDX1 positive cells. The terminaldifferentiation was induced by nicotinamide.

Small molecule inhibitors have also been used for induction ofpancreatic endocrine precursor cells. For example, small moleculeinhibitors of TGF-β receptor and BMP receptors (Development 2011,138:861-871; Diabetes 2011, 60:239-247) have been used to significantlyenhance the number of pancreatic endocrine cells. In addition, smallmolecule activators have also been used to generate definitive endodermcells or pancreatic precursor cells (Curr. Opin. Cell Biol. 2009,21:727-732; Nature Chem. Biol. 2009, 5:258-265).

HB9 (also known as HIXB9 and MNX1) is a basic helix-loop-helix (“bHLH”)transcriptional activator protein expressed early in pancreasdevelopment starting at approximately embryonic day eight. Expression ofHB9 is transient and peaks at about day 10.5 in pancreatic epithelium,being expressed in PDX1 and NKX6.1 expressing cells. At about day 12.5,HB9 expression declines and at later stages it becomes restricted onlyto β cells. In mice homozygous for a null mutation of HB9, the dorsallobe of the pancreas fails to develop (Nat. Genet. 23:67-70, 1999; Nat.Genet. 23:71-75, 1999). HB9-/β-cells express low levels of the glucosetransporter, GLUT2, and NKX6.1. Furthermore, HB9−/−pancreas shows asignificant reduction in the number of insulin positive cells while notsignificantly affecting expression of other pancreatic hormones. Thus,temporal control of HB9 is essential to normal β cell development andfunction. While not much is known about factors regulating HB9expression in β cells, a recent study in zebrafish suggests thatretinoic acid can positively regulate expression of HB9 (Development,138, 4597-4608, 2011).

In U.S. patent application Ser. No. 13/998,883, incorporated herein inits entirety by reference, it was demonstrated that triiodothyronine(“T3”) may act as an inducer of HB9 protein expression indifferentiating cells toward β cells. Methods for generating pancreaticendoderm cells that were positive for NKX6.1, PDX1 and HB9 using of oneor both of T3 and T4 are also disclosed therein. Additionally, and asdisclosed in U.S. patent application Ser. No. 13/998,884, incorporatedherein in its entirety by reference, it was demonstrated that expressionof pancreatic endocrine markers can be significantly enhanced byculturing at the air-liquid interface and using T3 and activinreceptor-like kinase (“ALK”) 5 inhibitors.

A variety of transcription factors regulate the differentiation ofpancreatic endocrine cells into insulin secreting β cells. Among thesefactors is v-mafavian musculoaponeurotic fibrosarcoma oncogene homolog A(“MAFA”). In fact, it is believed that MAFA may be a master regulator inβ cells of glucose stimulated insulin secretion.

In general, the process of differentiating progenitor cells tofunctional β cells goes through various stages and great strides havebeen made in improving protocols to generate pancreatic cells fromprogenitor cells, such as human pluripotent stem cells. Despite theseadvances in research, each step in the process of differentiatingprogenitor cells presents a unique challenge. As such, there is still aneed for a further differentiation protocol development for the purposeof producing functional endocrine cells and, in particular, functional βcells. In particular, it is desirable to develop processes in which theexpression of MAFA in pancreatic endocrine cells is enhanced.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A, 1B, 1C, 1D, 1E, 1F, 1G, 1H, 1I, 1J, 1K, 1L and 1M are graphsdepicting data from real-time PCR analyses of the fold change of geneexpression over undifferentiated ES cells of PDX1, NKX6.1, PAX4, PAX6,NGN3, MAFA, ABCC8, chromogranin-A, G6PC2, IAPP, insulin, glucagon andPTF1a from the stem cell line H1 differentiated in accordance withExample 1.

FIGS. 2A, 2B and 2C depict FACS profiles of Stage 3 cells,differentiated according to Example 1, and stained for: PDX1 (X-axis)co-stained with Ki67 (Y-axis) in FIG. 2A; PDX1 (X-axis) co-stained withCDX2 (Y-axis) in FIG. 2B; and NKX6.1 in FIG. 2C.

FIGS. 3A. 3B, 3C and 3D depict FACS profiles of Stage 4 cells,differentiated according to Example 1, and stained for: chromogranin(X-axis) co-stained with NKX6.1 (Y-axis) in FIG. 3A; PDX1 (X-axis)co-stained with Ki67 (Y-axis) in FIG. 3B; NKX6.1 (X-axis) co-stainedwith insulin (Y-axis) in FIG. 3C; and NeuroD1 in FIG. 3D.

FIGS. 4A, 4B, 4C, 4D and 4E depict FACS profiles of Stage 5 cells,differentiated according to Example 1, and stained for: chromogranin(X-axis) co-stained with NKX6.1 (Y-axis) in FIG. 4A; PDX1 (X-axis)co-stained with Ki67 (Y-axis) in FIG. 4B; and NKX6.1 (X-axis) co-stainedwith insulin (Y-axis) in FIG. 4C; NeuroD1 in FIG. 4D; and insulin(X-axis) co-stained with glucagon (Y-axis).

FIGS. 5A. 5B, 5C, 5D, 5E and 5F depict FACS profiles of Stage 6 cells,differentiated according to Example 1, and stained for: chromogranin(X-axis) co-stained with NKX6.1 (Y-axis) in FIG. 5A; PDX1 (X-axis)co-stained with Ki67 (Y-axis) in FIG. 5B; and NKX6.1 (X-axis) co-stainedwith insulin (Y-axis) in FIG. 5C; PAX6 (X-axis) co-stained with Oct 3/4(Y-axis) in FIG. 5D; insulin (X-axis) co-stained with glucagon (Y-axis)in FIG. 5E; and FOXA2 in FIG. 5F.

FIGS. 6A, 6B, 6C, 6D, 6E and 6F depict FACS profiles of Stage 7 cells,differentiated according to Example 1, and stained for: chromogranin(X-axis) co-stained with NKX6.1 (Y-axis) in FIG. 6A; PDX1 (X-axis)co-stained with Ki67 (Y-axis) in FIG. 6B; and NKX6.1 (X-axis) co-stainedwith insulin (Y-axis) in FIG. 6C; PAX6 (X-axis) co-stained with Oct 3/4(Y-axis) in FIG. 6D; insulin (X-axis) co-stained with glucagon (Y-axis)in FIG. 6E; and FOXA2 in FIG. 6F.

FIG. 7 is a graph of the percent expression of multiple pancreaticendoderm markers (FOXA2, PDX1, NKX6.1), an undifferentiated ES cellmarker (Oct3/4), endocrine markers (PAX6, IS1-1, NKX2.2, chromogranin),and hormone (insulin, glucagon) from Stage 3 through Stage 7 cellsdifferentiated according to Example 1.

FIGS. 8A, 8B. 8C, 8D and 8E are graphs depicting data from real-time PCRanalyses of the fold change of expression of insulin and MAFA ofdifferentiated cells over undifferentiated cells after treatment withsmall molecules in Stage 6-7.

FIG. 9 is graph depicting data from real-time PCR analyses of the foldchange of expression of AXL and GAS6 of differentiated cells of Example4 over undifferentiated cells.

FIGS. 10A, 10B, 10C, 10D, 10E and 10F are graphs of data from real-timePCR analyses of the fold change of expression of MAFA, UCN3, G6PC2,NKX6.1, PDX1 and insulin of differentiated cells over undifferentiatedcells after treatment with small molecules in Stage 7 in accordance withExample 6.

FIGS. 11A, 11B, 11C and 11D are graphs depicting data from real-time PCRanalyses of the fold change of expression of MAFA. PDX1, NKX6.1, andinsulin of differentiated cells over undifferentiated cells aftertreatment with small molecules in Stage 7 in accordance with Example 7

DETAILED DESCRIPTION OF THE INVENTION

The following detailed description of the invention will be betterunderstood when read in conjunction with the appended figures. For thepurpose of illustrating the invention, the figures demonstrateembodiments of the present invention. However, the invention is notlimited to the precise arrangements, examples, and instrumentalitiesshown. For clarity of disclosure, and not by way of limitation, thedetailed description of the invention is divided into subsections thatdescribe or illustrate certain features, embodiments, or applications ofthe present invention.

The present invention is directed to generating pancreatic endocrinecells of a more mature phenotype by treatment of less mature pancreaticendocrine cells with certain small molecules. In certain embodiments ofthe invention, pancreatic endocrine cells are cultured, in one or morestages, in the presence of small molecules that are one or more of aprotein methyltransferase inhibitor, an aurora kinase inhibitor, and ap90 ribosomal S6 kinase (“RSK”) inhibitor. Thus, the present inventionprovides cell cultures for differentiating pluripotent stem cells tocells exhibiting characteristics of pancreatic endocrine cells of amature phenotype, as well as differentiation media that initiates andfacilitates such differentiation, and differentiated cells and cellpopulations resulting from the differentiation. The methods of theinvention provide for the formation of a pancreatic endocrine cellpopulation wherein at least 10%, preferably at least 20%, morepreferably at least 30% and most preferably at least 50%, of the cellsexpress single hormonal insulin and are PDX1, NKX6.1 and MAFA positive.

Advantageously and preferably, the cell cultures and differentiationmedia of the invention may be used in conjunction with differentiationat the air-liquid interface. The culturing may occur at the air-liquidinterface for all stages involved in the differentiation pathway frompluripotent stem cells to pancreatic endocrine cells of a maturephenotype or it may involve culturing on a planar culture submersed inmedium for the early stages of differentiation followed by culturing atthe air-liquid interface during one or more of the later stages ofdifferentiation. More preferably, the processes of the inventioninvolves the combination of culturing pluripotent stem cells on asupport surface submerged in medium through the early stages, and thenculturing at the air-liquid interface for the later stages ofdifferentiation. In such embodiments, the cells may initially be seededon a solid surface for submerged culturing and then removed from thesolid support and re-seeded on a porous support for culturing at theair-liquid interface. Alternatively, the cells may be seeded initiallyon a porous support that is then submerged in media for the early stagesof differentiation and subsequently positioned at the air-liquidinterface for the later stages of differentiation.

In yet another embodiment, differentiation at one or more stages also iscarried out in the presence of one or more of T3, T4, analogues thereofand, optionally but preferably, with an activin receptor-like kinase 5(“ALK 5”) inhibitor. In a preferred embodiment, a population ofpancreatic endoderm/endocrine precursor cells are cultured in mediacontaining one or more of T3, T4, analogues thereof and an ALK 5inhibitor to pancreatic endocrine cells. In a more preferred embodiment,the resulting pancreatic endocrine cells are further differentiated inthe presence of media containing one or more of T3. T4, analoguesthereof and an ALK 5 inhibitor.

It is a particular discovery of the invention that treatment ofpancreatic endoderm cells with a combination of one or more of an ALK 5inhibitor and a thyroid receptor agonist followed by culturing of theresulting pancreatic endocrine cells in combination with one or more ofan inhibitor of protein methyltransferase DOT1L, an aurora kinaseinhibitor, and an RSK inhibitor significantly enhances the number ofcells in a population expressing, single hormonal insulin, MAFA, PDX1,and NKX6.1 as well as increases the level of MAFA expression in thecells. The invention finds particular utility when used in conjunctionwith differentiation at the air-liquid interface.

DEFINITIONS

Stem cells are undifferentiated cells defined by their ability, at thesingle cell level, to both self-renew and differentiate. Stem cells mayproduce progeny cells, including self-renewing progenitors, non-renewingprogenitors, and terminally differentiated cells. Stem cells are alsocharacterized by their ability to differentiate in vitro into functionalcells of various cell lineages from multiple germ layers (endoderm,mesoderm, and ectoderm). Stem cells also give rise to tissues ofmultiple germ layers following transplantation and contributesubstantially to most, if not all, tissues following injection intoblastocysts.

Stem cells are classified by their developmental potential. Pluripotentstem cells are able to give rise to all embryonic cell types.

Differentiation is the process by which an unspecialized (“uncommitted”)or less specialized cell acquires the features of a specialized cell,for example a nerve cell or a muscle cell. A differentiated cell is onethat has taken on a more specialized (“committed”) position within thelineage of a cell. The term “committed”, when applied to the process ofdifferentiation, refers to a cell that has proceeded in thedifferentiation pathway to a point where, under normal circumstances, itwill continue to differentiate into a specific cell type or subset ofcell types, and cannot, under normal circumstances, differentiate into adifferent cell type or revert to a less differentiated cell type.“De-differentiation” refers to the process by which a cell reverts to aless specialized (or committed) position within the lineage of a cell.As used herein, the lineage of a cell defines the heredity of the cell,i.e., which cells it came from and to what cells it can give rise. Thelineage of a cell places the cell within a hereditary scheme ofdevelopment and differentiation. A lineage-specific marker refers to acharacteristic specifically associated with the phenotype of cells of alineage of interest and can be used to assess the differentiation of anuncommitted cell to the lineage of interest.

“Markers”, as used herein, are nucleic acid or polypeptide moleculesthat are differentially expressed in a cell of interest. In thiscontext, differential expression means an increased level for a positivemarker and a decreased level for a negative marker as compared to anundifferentiated cell or a cell at another stage of differentiation. Thedetectable level of the marker nucleic acid or polypeptide issufficiently higher or lower in the cells of interest compared to othercells, such that the cell of interest can be identified anddistinguished from other cells using any of a variety of methods knownin the art.

As used herein, a cell is “positive for” a specific marker, “positive”,or “+” when the specific marker is sufficiently detected in the cell.Similarly, the cell is “negative for”, “negative” or “−” for a specificmarker when the specific marker is not sufficiently detected in thecell. In particular, positive by fluorescence activated cell sortingcytometry (“FACS”) is usually greater than about 2%, whereas thenegative threshold by FACS is usually less than about 1%. Positive bypolymerase chain reaction cytometry (“PCR”) is usually less than orequal to about 30 cycles (Cts); whereas negative by PCR is usually morethan about 31 cycles.

In attempts to replicate the differentiation of pluripotent stem cellsinto functional pancreatic endocrine cells in static in vitro cellcultures, the differentiation process is often viewed as progressingthrough a number of consecutive stages. In particular, thedifferentiation process is commonly viewed as progressing throughmultiple stages. In this step-wise differentiation, “Stage 1” refers tothe first step in the differentiation process, the differentiation ofpluripotent stem cells into cells expressing markers characteristic ofthe definitive endoderm (“Stage 1 cells”). “Stage 2” refers to thesecond step, the differentiation of cells expressing markerscharacteristic of the definitive endoderm cells into cells expressingmarkers characteristic of gut tube cells (“Stage 2 cells”). “Stage 3”refers to the third step, differentiation of cells expressing markerscharacteristic of gut tube cells into cells expressing markerscharacteristic of foregut endoderm cells (“Stage 3 cells”). “Stage 4”refers to the fourth step, the differentiation of cells expressingmarkers characteristic of foregut endoderm cells into cells expressingmarkers characteristic of pancreatic foregut precursor cells (“Stage 4cells”). “Stage 5” refers to the fifth step, the differentiation ofcells expressing markers characteristic of pancreatic foregut precursorcells into cells expressing markers characteristic of one or both ofpancreatic endoderm cells and pancreatic endocrine precursor cells(collectively referred to as “Stage 5 cells” or, alternatively,“pancreatic endoderm/endocrine precursor cells”). Stage 6 refers to thesixth step, the differentiation of cells expressing markerscharacteristic of pancreatic endoderm/endocrine precursor cells intocells expressing markers characteristic of pancreatic endocrine cellsthat are immature beta cell (“Stage 6 cells”). Stage 6 cells expresssingle hormonal insulin and are PDX1, NKX6.1 and chromogranin positive.In the process of, and for purposes of producing the populations of andcells of the invention, a seventh step, “Stage 7”, is used and refers todifferentiation of cells expressing markers characteristic of pancreaticendocrine cells that are immature beta cells into cells expressingmarkers characteristic of pancreatic endocrine cells that are maturingbeta cells and that have a more mature phenotype as compared to Stage 6cells. By or “Stage 7 cells” is meant a pancreatic endocrine cell thatis single hormonal insulin +, MAFA+, NKX6.1+, and PDX1+ but alsoexpresses MAFA at a higher level than an immature beta cell.Additionally, the cell population resulting from carrying out Stage 7has a higher percentage of MAFA positive and single hormonal insulinexpressing cells as compared to populations of cells of Stage 6.

It is to be noted that not all cells in a particular population progressthrough these stages at the same rate. Consequently, it is not uncommonin in vitro cell cultures to detect the presence of cells that haveprogressed less, or more, down the differentiation pathways than themajority of cells present in the population, particularly at the laterdifferentiation stages. For example, it is not uncommon to see theappearance of markers characteristic of pancreatic endocrine cellsduring the culture of cells at Stage 5. For purposes of illustrating thepresent invention, characteristics of the various cell types associatedwith the above-identified stages are described herein.

“Definitive endoderm” as used herein, refers to cells which bear thecharacteristics of cells arising from the epiblast during gastrulationand which form the gastrointestinal tract and its derivatives.Definitive endoderm cells express at least one of the following markers:FOXA2 (also known as hepatocyte nuclear factor 3-β (“HNF3-β”)), GATA4,SOX17, CXCR4, Brachyury, Cerberus, OTX2, goosecoid, C-Kit, CD99, andMIXL1. Markers characteristic of the definitive endoderm cells areCXCR4, FOXA2, and SOX17. Thus, definitive endoderm cells may becharacterized by their expression of CXCR4, FOXA2, and SOX17. Inaddition, depending on the length of time cells are allowed to remain inStage 1, an increase in HNF4α may be observed.

“Gut tube cells”, as used herein, refers to cells derived fromdefinitive endoderm and that can give rise to all endodermal organs,such as lungs, liver, pancreas, stomach, and intestine. Gut tube cellsmay be characterized by their substantially increased expression ofHNF4α over that expressed by definitive endoderm cells. For example, aten- to forty-fold increase in mRNA expression of HNF4α may be observedduring Stage 2.

“Foregut endoderm cells”, as used herein, refers to cells that give riseto the esophagus, lungs, stomach, liver, pancreas, gall bladder, and aportion of the duodenum. Foregut endoderm cells express at least one ofthe following markers: PDX1, FOXA2, CDX2, SOX2, and HNF4α. Foregutendoderm cells may be characterized by an increase in expression of PDX1compared to gut tube cells. For example, greater than fifty percent ofthe cells in Stage 3 cultures typically express PDX1.

“Pancreatic foregut precursor cells”, as used herein, refers to cellsthat express at least one of the following markers: PDX1, NKX6.1, HNF6,NGN3, SOX9, PAX4, PAX6, ISL1, gastrin, FOXA2, PTF1a, PROX1 and HNF4α.Pancreatic foregut precursor cells may be characterized by beingpositive for the expression of at least one of PDX1, NKX6.1, and SOX9.

“Pancreatic endoderm cells”, as used herein, refers to cells thatexpress at least one of the following markers: PDX1, NKX6.1, HNF1β,PTF1α, HNF6, HNF4α, SOX9. NGN3, gastrin; HB9, or PROX1. Pancreaticendoderm cells may be characterized by their lack of substantialexpression of CDX2 or SOX2.

“Pancreatic endocrine precursor cells”, as used herein, refers topancreatic endoderm cells capable of becoming a pancreatic hormoneexpressing cell. Pancreatic endocrine precursor cells express at leastone of the following markers: NGN3; NKX2.2; NeuroD11; ISL1; PAX4; PAX6;or ARX. Pancreatic endocrine precursor cells may be characterized bytheir expression of NKX2.2 and NeuroD11.

“Pancreatic endocrine cells”, as used herein, refer to cells capable ofexpressing at least one of the following hormones: insulin, glucagon,somatostatin, ghrelin, and pancreatic polypeptide. In addition to thesehormones, markers characteristic of pancreatic endocrine cells includeone or more of NeuroD1, ISL1, PDX1, NKX6.1, ARX, NKX2.2, HB9 and PAX6.One subset of pancreatic endocrine cells is “immature beta cells” thatare cells capable of expressing insulin, but not glucagon, somatostatin,ghrelin, and pancreatic polypeptide. In addition, markers characteristicof immature beta cells include one or more of NeuroD1, ISL1, PDX1,NKX6.1, NKX2.2, HB9 and PAX6. A second subset of pancreatic endocrinecells is “maturing beta cells” that are cells capable of expressinginsulin, but not glucagon, somatostatin, ghrelin, and pancreaticpolypeptide. Additionally, markers characteristic of maturing beta cellsinclude one or more of NeuroD1, ISL1, PDX1, NKX6.1, NKX2.2, HB9, PAX6and MAFA. Yet another subset of pancreatic endocrine cells are thoseexpressing markers characteristic of mature beta cells and that can becharacterized by their expression of PDX1, NKX2.2, NKX6.1, NeuroD1,ISL1, HNF3β, HB9, MAFA and PAX6 along with an insulin release inresponse to a glucose challenge that is robust and increased incomparison to that of less mature beta cells.

“Air-liquid interface” or “ALI”, as used herein, refers to theair-liquid interface that exists in an open culture vessel or a culturevessel partially filled with medium. Although referred to herein as“air” for convenience, the invention is not limited to the mixture ofgases and compositions found in the ambient environment. The inventionspecifically contemplates and includes gaseous mixtures havingcompositions different from the ambient environment including, forexample, mixtures enriched for a particular component or in which aparticular component has been depleted or eliminated.

Used interchangeably herein are “d1”, “1d”, and “day 1”; “d2”, “2d”, and“day 2”, and so on. These number letter combinations refer to a specificday of incubation in the different stages during the stepwisedifferentiation protocol of the instant application.

“LDN-193189” refers to((6-(4-(2-(piperidin-1-yl)ethoxy)phenyl)-3-(pyridin-4-yl)pyrazolo[1,5-a]pyrimidine,hydrochloride)) a BMP receptor inhibitor available from ShanghaiChemPartner, Co., LTD.

Characterization, Source, Expansion and Culture of Pluripotent StemCells A. Characterization of Pluripotent Stem Cells

Pluripotent stem cells may express one or more of the designatedTRA-1-60 and TRA-1-81 antibodies (Thomson et al. 1998, Science282:1145-1147). Differentiation of pluripotent stem cells in vitroresults in the loss of TRA-1-60 and TRA-1-81 expression.Undifferentiated pluripotent stem cells typically have alkalinephosphatase activity, which can be detected by fixing the cells with 4%paraformaldehyde, and then developing with an alkaline phosphatasesubstrate kit sold under the trademark VECTOR® Red, as described by themanufacturer (Vector Laboratories, Inc., Burlingame, Calif.).Undifferentiated pluripotent stem cells also typically express OCT4 andTERT, as detected by reverse transcription polymerase chain reaction(“RT-PCR”).

Another desirable phenotype of propagated pluripotent stem cells is apotential to differentiate into cells of all three germinal layers:endoderm, mesoderm, and ectoderm. Pluripotency of stem cells may beconfirmed, for example, by injecting cells into severe combinedimmunodeficiency (“SCID”) mice, fixing the teratomas that form using 4%paraformaldehyde, and then examining histologically for evidence of celltypes from these three germ layers. Alternatively, pluripotency may bedetermined by the creation of embryoid bodies and assessing the embryoidbodies for the presence of markers associated with the three germinallayers.

Propagated pluripotent stem cell lines may be karyotyped using astandard G-banding technique and compared to published karyotypes of thecorresponding primate species. It is desirable to obtain cells that havea “normal karyotype,” which means that the cells are euploid, whereinall human chromosomes are present and not noticeably altered.

B. Sources of Pluripotent Stem Cells

Any pluripotent stem cells may be used in the methods of the invention.Exemplary types of pluripotent stem cells that may be used includeestablished lines of pluripotent cells, including pre-embryonic tissue(such as, a blastocyst), embryonic tissue, or fetal tissue taken anytime during gestation, typically but not necessarily, beforeapproximately 10 to 12 weeks gestation. Non-limiting examples areestablished lines of human embryonic stem cells (“hESCs”) or humanembryonic germ cells, such as, the human embryonic stem cell lines H1(NIH Code: WA01), H7 (NIH Code: WA07), H9 (NIH Code: WA09) (WiCellResearch Institute, Madison, Wis., USA), and SA002 (Cellartis ABCorporation, Goteburg, Sweden).

Cells taken from a pluripotent stem cell population already cultured inthe absence of feeder cells are also suitable. Induced pluripotent cells(IPS), or reprogrammed pluripotent cells, derived from adult somaticcells using forced expression of a number of pluripotent relatedtranscription factors, such as OCT4, NANOG, SOX2, KLF4, and ZFP42 (AnnuRev Genomics Hum Genet 2011, 12:165-185; see also IPS, Cell, 126(4):663-676) may also be used. The human embryonic stem cells used in themethods of the invention may also be prepared as described by Thomson etal. (U.S. Pat. No. 5,843,780; Science, 1998, 282:1145-1147; Curr Top DevBiol 1998, 38:133-165; Proc Natl Acad Sci U.S.A. 1995, 92:7844-7848).Mutant human embryonic stem cell lines, such as, BG01v (BresaGen,Athens, Ga.), or cells derived from adult human somatic cells, such as,cells disclosed in Takahashi et al., Cell 131: 1-12 (2007) may also beused. In certain embodiments, pluripotent stem cells suitable for use inthe present invention may be derived according to the methods describedin: Li et al. (Cell Stem Cell 4: 16-19, 2009); Maherali et al. (CellStem Cell 1: 55-70, 2007); Stadtfeld et al. (Cell Stem Cell 2: 230-240);Nakagawa et al. (Nature Biotechnol 26: 101-106, 2008); Takahashi et al.(Cell 131: 861-872, 2007); and U.S. Patent App. Pub. No. 2011/0104805.In certain embodiments, pluripotent stem cells suitable for use in thepresent invention may be considered “naïve” and derived according to themethods described in: Gafni et al. (Nature, 504:282, 2013), and Ware etal. (PNAS, 111: 4484-4489, 2014). All of these references, patents, andpatent applications are herein incorporated by reference in theirentirety, in particular, as they pertain to the isolation, culture,expansion and differentiation of pluripotent cells.

Other sources of pluripotent stem cells include induced pluripotent stemcells (IPS, Cell, 126(4): 663-676). Yet other sources of suitable cellsinclude human umbilical cord tissue-derived cells, human amnioticfluid-derived cells, human placental-derived cells, and humanparthenotes. In one embodiment, the umbilical cord tissue-derived cellsmay be obtained by the method of U.S. Pat. No. 7,510,873. In anotherembodiment, the placental tissue-derived cells may be obtained using themethods of U.S. Patent Application Publication No. 2005/0058631. Inanother embodiment, the amniotic fluid-derived cells may be obtainedusing the methods of U.S. Patent App. Pub. No. 2007/0122903. Thedisclosure of each of these patent applications is incorporated in itsentirety herein as it pertains to the isolation and characterization ofthe cells. In certain embodiments, the pluripotent stem cells may be ofnon-embryonic origins.

C. Expansion and Culture of Pluripotent Stem Cells

Pluripotent stem cells are typically cultured on a layer of feeder cellsthat support the pluripotent stem cells in various ways. Alternatively,pluripotent stem cells may be cultured in a culture system that isessentially free of feeder cells, but nonetheless supports proliferationof pluripotent stem cells without undergoing substantialdifferentiation. The growth of pluripotent stem cells in feeder-freeculture without differentiation is often supported using a mediumconditioned by culturing previously with another cell type.Alternatively, the growth of pluripotent stem cells in feeder-freeculture without differentiation can be supported using a chemicallydefined medium.

Pluripotent cells may be readily expanded in culture using variousfeeder layers or by using matrix protein coated vessels. Alternatively,chemically defined surfaces in combination with defined media, such asmedia sold under the trademark mTESR®1 (StemCell Technologies, Inc.,Vancouver, B.C., Canada), may be used for routine expansion of thecells. Pluripotent cells may be readily removed from culture platesusing enzymatic digestion, mechanical separation, or various calciumchelators such as ethylenediaminetetraacetic acid (“EDTA”).Alternatively, pluripotent cells may be expanded in suspension in theabsence of any matrix proteins or feeder layer.

Many different known methods of expanding and culturing pluripotent stemcells may be used in the claimed invention. For example, the methods ofthe invention may use the methods of Reubinoff et al., Thompson et al.,Richards et al. and U.S. Patent App. Pub. No. 2002/0072117. Reubinoff etal. (Nature Biotechnology 18: 399-404 (2000)) and Thompson et al.(Science 282: 1145-1147 (1998)) disclose the culture of pluripotent stemcell lines from human blastocysts using a mouse embryonic fibroblastfeeder cell layer. Richards et al. (Stem Cells 21: 546-556, 2003)evaluated a panel of eleven different human adult, fetal, and neonatalfeeder cell layers for their ability to support human pluripotent stemcell culture, noting that “human embryonic stem cell lines cultured onadult skin fibroblast feeders retain human embryonic stem cellmorphology and remain pluripotent.” U.S. Patent App. Pub. No.2002/0072117 discloses cell lines that produce media that support thegrowth of primate pluripotent stem cells in feeder-free culture. Thecell lines employed are mesenchymal and fibroblast-like cell linesobtained from embryonic tissue or differentiated from embryonic stemcells. U.S. Patent App. Pub. No. 2002/072117 also discloses the use ofthe cell lines as a primary feeder cell layer.

Other suitable known methods of expanding and culturing pluripotent stemcells are disclosed, for example, in Wang et al., Stojkovic et al.,Miyamoo et al. and Amit et al. Wang et al. (Stem Cells 23: 1221-1227,2005) disclose methods for the long-term growth of human pluripotentstem cells on feeder cell layers derived from human embryonic stemcells. Stojkovic et al. (Stem Cells 2005 23: 306-314, 2005) disclose afeeder cell system derived from the spontaneous differentiation of humanembryonic stem cells. Miyamoto et al. (Stem Cells 22: 433-440, 2004)disclose a source of feeder cells obtained from human placenta. Amit etal. (Biol. Reprod 68: 2150-2156, 2003) disclose a feeder cell layerderived from human foreskin.

Other suitable methods of expanding and culturing pluripotent stem cellsare disclosed, for example, in Inzunza et al., U.S. Pat. No. 6,642,048,WO 2005/014799, Xu et al. and U.S. Patent App. Pub. No. 2007/0010011.Inzunza et al. (Stem Cells 23: 544-549, 2005) disclose a feeder celllayer from human postnatal foreskin fibroblasts. U.S. Pat. No. 6,642,048discloses media that support the growth of primate pluripotent stemcells in feeder-free culture, and cell lines useful for production ofsuch media. U.S. Pat. No. 6,642,048 reports mesenchymal andfibroblast-like cell lines obtained from embryonic tissue ordifferentiated from embryonic stem cells, as well as methods forderiving such cell lines, processing media, and growing stem cells usingsuch media. WO 2005/014799 discloses a conditioned medium for themaintenance, proliferation, and differentiation of mammalian cells. WO2005/014799 reports that the culture medium produced via the disclosureis conditioned by the cell secretion activity of murine cells; inparticular, those differentiated and immortalized transgenichepatocytes, named MMH (Met Murine Hepatocyte). Xu et al. (Stem Cells22: 972-980, 2004) discloses a conditioned medium obtained from humanembryonic stem cell derivatives that have been genetically modified toover express human telomerase reverse transcriptase. U.S. Patent App.Pub. No. 2007/0010011 discloses a chemically defined culture medium forthe maintenance of pluripotent stem cells.

A known alternative culture system employs serum-free mediumsupplemented with growth factors capable of promoting the proliferationof embryonic stem cells. Examples of such culture systems include, butare not limited, to Cheon et al., Levenstein et al. and U.S. Patent App.Pub. No. 2005/0148070. Cheon et al. (BioReprod DOI:10.1095/biolreprod.105.046870, Oct. 19, 2005) disclose a feeder-free, serum-free culturesystem in which embryonic stem cells are maintained in unconditionedserum replacement medium supplemented with different growth factorscapable of triggering embryonic stem cell self-renewal. Levenstein etal. (Stem Cells 24: 568-574, 2006) disclose methods for the long-termculture of human embryonic stem cells in the absence of fibroblasts orconditioned medium, using media supplemented with bFGF. U.S. Patent App.Pub. No. 2005/0148070 discloses a method of culturing human embryonicstem cells in defined media without serum and without fibroblast feedercells, the method comprising: culturing the stem cells in a culturemedium containing albumin, amino acids, vitamins, minerals, at least onetransferrin or transferrin substitute, at least one insulin or insulinsubstitute, the culture medium essentially free of mammalian fetal serumand containing at least about 100 ng/ml of a fibroblast growth factorcapable of activating a fibroblast growth factor signaling receptor,wherein the growth factor is supplied from a source other than just afibroblast feeder layer, the medium supported the proliferation of stemcells in an undifferentiated state without feeder cells or conditionedmedium.

Still other known suitable methods of culturing and expandingpluripotent stem cells are disclosed in U.S. Patent App. Pub. No.2005/0233446, U.S. Pat. No. 6,800,480, U.S. Patent App. Pub. No.2005/0244962 and WO 2005/065354. U.S. Patent App. Pub. No. 2005/0233446discloses a defined media useful in culturing stem cells, includingundifferentiated primate primordial stem cells. In solution, the mediais substantially isotonic as compared to the stem cells being cultured.In a given culture, the particular medium is a base medium and an amountof each of bFGF, insulin, and ascorbic acid necessary to supportsubstantially undifferentiated growth of the primordial stem cells. U.S.Pat. No. 6,800,480 reports that a cell culture medium for growingprimate-derived primordial stem cells in a substantiallyundifferentiated state is provided which includes a low osmoticpressure, low endotoxin basic medium that is effective to support thegrowth of primate-derived primordial stem cells. The disclosure of theU.S. Pat. No. 6,800,480 patent further reports that the basic medium iscombined with a nutrient serum effective to support the growth ofprimate-derived primordial stem cells and a substrate selected fromfeeder cells and an extracellular matrix component derived from feedercells. This medium is further noted to include non-essential aminoacids, an anti-oxidant, and a first growth factor selected fromnucleosides and a pyruvate salt. U.S. Patent App. Pub. No. 2005/0244962reports that one aspect of the disclosure provides a method of culturingprimate embryonic stem cells and that the stem cells in culture areessentially free of mammalian fetal serum (preferably also essentiallyfree of any animal serum) and in the presence of fibroblast growthfactor that is supplied from a source other than just a fibroblastfeeder layer.

WO 2005/065354 discloses a defined, an isotonic culture medium that isessentially feeder-free and serum-free, that is a basal medium, bFGF,insulin and ascorbic acid in amounts sufficient to support growth ofsubstantially undifferentiated mammalian stem cells. Furthermore, WO2005/086845 discloses a method for maintenance of an undifferentiatedstem cell, said method comprising exposing a stem cell to a member ofthe transforming growth factor-beta (“TGF-β”) family of proteins, amember of the fibroblast growth factor (“FGF”) family of proteins, ornicotinamide in an amount sufficient to maintain the cell in anundifferentiated state for a sufficient amount of time to achieve adesired result.

The pluripotent stem cells may be plated onto a suitable culturesubstrate. In one embodiment, the suitable culture substrate is anextracellular matrix component, such as those derived from basementmembrane or that may form part of adhesion molecule receptor-ligandcouplings. A suitable culture substrate is a reconstituted basementmembrane sold under the trademark MATRIGEL™ (Corning Incorporated,Corning, N.Y.). MATRIGEL™ is a soluble preparation from Engelbreth-HolmSwarm tumor cells that gels at room temperature to form a reconstitutedbasement membrane.

Other extracellular matrix components and component mixtures known inthe art are suitable as an alternative. Depending on the cell type beingproliferated, this may include laminin, fibronectin, proteoglycan,entactin, heparin sulfate, and the like, alone or in variouscombinations.

The pluripotent stem cells may be plated onto the substrate in asuitable distribution and in the presence of a medium, that promotescell survival, propagation, and retention of the desirablecharacteristics. All these characteristics benefit from carefulattention to the seeding distribution and can readily be determined byone of skill in the art. Suitable culture media may be made from thefollowing components, Dulbecco's Modified Eagle's medium (“DMEM”) soldunder the trademark GIBCO® (Catalog No. 11965-092) by Life TechnologiesCorporation, Grand Island New York; Knockout Dulbecco's Modified Eagle'smedium (“KO DMEM”) sold under the trademark GIBCO® (Catalog No.10829-018) by Life Technologies Corporation; Ham's F12/50% DMEM basalmedium; 200 mM L-glutamine sold under the trademark GIBCO® (Catalog No.25030-081) by Life Technologies; non-essential amino acid solution soldunder the trademark GIBCO® (Catalog No. 11140-050) by Life Technologies;β-mercaptoethanol, Sigma-Aldrich Company, LLC Saint Louis, Mo., (CatalogNo. M7522); human recombinant basic fibroblast growth factor (“bFGF”)sold under the trademark GIBCO® (Catalog No. 13256-029) by LifeTechnologies.

Large-scale expansion and controlled differentiation processes of humanembryonic stem cells can also be achieved using suspension bioreactors.Such systems may be able to generate clinically relevant cell numberswith greater efficacy in a controlled culture system. It is known to useestablished bioreactor culture systems that allow for the expansion ofpluripotent murine and hES cells for example as disclosed in Journal ofBiotechnology, May 2014, Vol. 178: 54-64, Stem Cell Reports, April 2014,Vol. 3, No. 6:1132, and Tissue Engineering Part C: Methods, February2013, Vol. 19, No. 2: 166-180.

Differentiation of Pluripotent Stem Cells

As pluripotent cells differentiate towards β cells, they differentiatethrough various stages each of which may be characterized by thepresence or absence of particular markers. Differentiation of the cellsinto these stages is achieved by the specific culturing conditionsincluding the presence and lack of certain factors added to the culturemedia. In general, this differentiation may involve differentiation ofpluripotent stem cells into definitive endoderm lineage, and definitiveendoderm, cells. These cells may then be further differentiated into guttube cells, which in turn may then be differentiated into foregutendoderm cells. Foregut endoderm cells may be differentiated intopancreatic foregut precursor cells which may then be furtherdifferentiated into pancreatic endoderm cells, pancreatic endocrineprecursor cells or both. These cells may be differentiated intopancreatic hormone producing or secreting cells. This applicationprovides for the staged differentiation of pluripotent stem cellstowards the pancreatic endocrine cells, preferably by culturing thecells at the air-liquid interface that exists within a culture vesselpartially filled with medium, specifically by culturing cells at theair-liquid interface in one or more of Stages 5 through 7.

One or more of the thyroid hormones triiodothyronine (“T3”) andthyroxine (“T4”), and analogues thereof, alone or in further combinationwith an ALK 5 inhibitor may be used in the cell culturing at one or moreof Stages 1 through 7 of differentiation, and preferably at each ofStages 5 through 7. Alternatively, the ALK 5 inhibitor may be used alonein one or more stages of differentiation, but preferably at each ofStages 5 through 7. More preferably, one or more of the thyroid hormonesor their analogues and an ALK 5 inhibitor is used in one or moredifferentiation stages and preferably at each of Stages 5 through 7.Suitable thyroid hormone analogues may include, without limitation: GC-1(Sobertirome) (available from R&D Systems, Inc. Minneapolis, Minn.);3,5-diiodothryopropionic acid (“DIPTA”); KB-141 discussed in J. SteroidBiochem. Mol. Biol., 2008, 111: 262-267 and Proc. Natl. Acad. Sci. US2003, 100: 10067-10072; MB07344 discussed in Proc. Natl. Acad. Sci. US2007, 104: 15490-15495; T0681 discussed in J. Lipid Res., May 2009,50:938 and Endocr. Pract. 2012, 18(6): 954-964, the disclosures of whichare incorporated herein by reference in their entireties. Useful ALK5inhibitors include: ALK5 inhibitor II (Enzo Life Sciences, Inc.,Farmingdale, N.Y.), which is also the preferred ALK5 inhibitor; ALK5i(Axxora, Inc., San Diego, Calif.), SD208 (R&D Systems); TGF-β inhibitorSB431542 (Xcess Biosciences. Inc., San Diego, Calif.); ITD-1 (XcessBiosciences); LY2109761 (Xcess Biosciences); A83-01 (Xcess Biosciences);LY2157299 (Xcess Biosciences); TGF-β receptor inh V (EMD MilliporeChemical, Gibstown, N.J.); TGF-β receptor inh I (EMD Millipore); TGF-βreceptor inh IV (EMD Millipore); TGF-β receptor inh VII (EMD Millipore);TGF-β receptor inh VIII (EMD Millipore); TGF-β receptor inh II (EMDMillipore); TGF-β receptor inh VI (EMD Millipore); and TGF-β receptorinh VI (EMD Millipore).

In addition, in preferred embodiments of the invention, the methodsinclude treating cells at one or more stages, but preferably treatingcells during Stage 7, with a differentiation medium that includes one orboth of an antioxidant, such as vitamin E, acetyl cysteine, vitamin C,Antioxidant Supplement (Catalog No. A1345, Sigma-Aldrich Company, LLCSaint Louis, Mo.), glutathione, superoxide dismutase, catalase and thelike and combinations thereof. In still more preferred embodiments, incarrying out Stage 6, a gamma secretase inhibitor is used, which can begamma secretase inhibitor XX (EMD Millipore), gamma secretase inhibitorXXI (EMD Millipore), gamma secretase inhibitor XVI (EMD Millipore),N-[(3,5-difluorophenyl)acetyl]-L-alanyl-2-phenyl]glycine-1,1-dimethylethylester (“DAPT”) (Catalog. Ni. 2634, Tocris Bioscience, Bristol, UnitedKingdom), and the like and combinations thereof. Useful amounts of gammasecretase inhibitor may be about 50 nM to 5000 nM, preferably about 50nM to 500 nM. The amount of antioxidant may be about 0.1 to 100 μM,alternatively about 0.1 to 20 μM, and preferably about 1 to 10 μM.Alternatively, useful amounts of antioxidant may be about 100 nM to 5mM, about 1000 nM to 2 mM, and preferably about 0.1 to 1 mM.

In most preferred embodiments of the invention, certain small moleculesare used in the medium of one or more stages of differentiation,preferably at one or both of Stages 6 and 7. The small molecules ofinterest are those capable of inhibiting aurora kinase, p90 ribosomal S6kinase, or methyl transferase DOT1L and preferably are used along withantioxidants that reduce oxidative stress of cultured cells. Useful suchinhibitors include aurora kinase inhibitor II(4-(4′-benzamidoanilino)-6,7-dimethoxyquinazoline), SNS 314 mesylate(N-(3-Chlorophenyl)-N′-[5-[2-(thieno[3,2-d]pyrimidin-4-ylamino)ethyl]-2-thiazolyl]ureamethanesulfonate), GSK1070916(3-(4-(4-(2-(3-((dimethylamino)methyl)phenyl)-1H-pyrrolo[2,3-b]pyridin-4-yl)-1-ethyl-1H-pyrazol-3-yl)phenyl)-1,1-dimethylurea),TAK-901(5-(3-(ethylsulfonyl)phenyl)-3,8-dimethyl-N-(1-methylpiperidin-4-yl)-9H-pyrido[2,3-b]indole-7-carboxamide),RSK inhibitor II (a racemic mixture of dihydropteridinone2-(3,5-difluoro-4-hydroxy-anilino)-8-isopentyl-5,7-dimethyl-7H-pteridin-6-one),and EPZ-5676 (9H-Purin-6-amine,9-[5-deoxy-5-[[cis-3-[2-[6-(1,1-dimethylethyl)-1H-benzimidazol-2-yl]ethyl]cyclobutyl](1-methylethyl)amino]-β-D-ribofuranosyl]-),and combinations thereof. Of particular interest are aurora kinaseinhibitor II, and RSK inhibitor II, and an inhibitor of DOT1L,particularly EPZ-5676. In a preferred embodiment of the invention, thesmall molecule is used in the medium of one or more of Stage 6 and 7 andmore preferably in Stage 7. The amount of small molecule useful may bedetermined by selecting the amount showing the best expression ofmaturation markers and which amounts are not producing toxic effects.Typically, the amounts useful will be about 500 nM to 10 μM,alternatively, about 500 nM to 5 μM, and preferably about 500 nM to 2μM.

Differentiation of Pluripotent Cells into Cells Expressing MarkersCharacteristic of Pancreatic Endocrine Cells with a Mature Phenotype

Characteristics of pluripotent stem cells are well known to thoseskilled in the art, and additional characteristics of pluripotent stemcells continue to be identified. Pluripotent stem cell markers include,for example, the expression of one or more of the following: ABCG2,cripto, FOXD3, CONNEXIN43, CONNEXIN45, OCT4, SOX2, NANOG, hTERT, UTF1,ZFP42, SSEA-3, SSEA-4, TRA-1-60, TRA-1-81. These may be detectable byRT-PCR.

Exemplary pluripotent stem cells include the human embryonic stem cellline H9 (NIH code: WA09), the human embryonic stem cell line H1 (NIHcode: WA01), the human embryonic stem cell line H7 (NIH code: WA07), andthe human embryonic stem cell line SA002. Also suitable are cells thatexpress at least one of the following markers characteristic ofpluripotent cells: ABCG2, cripto, CD9, FOXD3, CONNEXIN43, CONNEXIN45,OCT4, SOX2, NANOG, hTERT, UTF1, ZFP42, SSEA-3, SSEA-4, TRA-1-60, andTRA-1-81.

Suitable for use in the present invention is a cell that expresses atleast one of the markers characteristic of the definitive endodermlineage. In one aspect of the present invention, a cell expressingmarkers characteristic of the definitive endoderm lineage is a primitivestreak precursor cell. In an alternate aspect, a cell expressing markerscharacteristic of the definitive endoderm lineage is a mesendoderm cell.In an alternate aspect, a cell expressing markers characteristic of thedefinitive endoderm lineage is a definitive endoderm cell.

Also suitable for use in the present invention is a cell that expressesat least one of the markers characteristic of the pancreatic endodermlineage. In one aspect of the present invention, a cell expressingmarkers characteristic of the pancreatic endoderm lineage is apancreatic endoderm cell wherein the expression of PDX1 and NKX6.1 aresubstantially higher than the expression of CDX2 and SOX2. In certainembodiments, more than 30% of the cells express PDX1 and NKX6.1 and lessthan 30% of the cells express CDX2 or SOX2 as measured by FACS.Particularly useful are cells in which the expression of PDX1 and NKX6.1is at least two-fold higher than the expression of CDX2 or SOX2.

Still also suitable for use in the present invention is a cell thatexpresses at least one of the markers characteristic of the pancreaticendocrine lineage. In one aspect of the invention, a cell expressingmarkers characteristic of the pancreatic endocrine lineage is apancreatic endocrine cell. The pancreatic endocrine cell may be apancreatic hormone-expressing cell meaning a cell capable of expressingat least one of the following hormones: insulin, glucagon, somatostatin,ghrelin, or pancreatic polypeptide. In a preferred embodiment, thepancreatic endocrine cell is an insulin-producing β cell.

In certain embodiments of the invention, to arrive at the cellsexpressing markers characteristic of the pancreatic endocrine beta cellsof a mature phenotype, a protocol starting with pluripotent stem cellsis employed. This protocol includes:

-   -   Stage 1: Pluripotent stem cells such as embryonic stem cells        obtained from cell culture lines are treated with the        appropriate factors to induce formation of definitive endoderm        cells.    -   Stage 2: Cells resulting from Stage 1 are treated with the        appropriate factors to induce formation of cells into markers        expressing characteristic of gut tube cells.    -   Stage 3: Cells resulting from Stage 2 cells are treated with the        appropriate factors to induce further differentiation into cells        expressing markers characteristic of foregut endoderm cells.    -   Stage 4: Cells resulting from Stage 3 are treated with the        appropriate factors to induce further differentiation into cells        expressing markers characteristic of pancreatic foregut        precursor cells. The cells are optionally cultured at the        air-liquid interface at late Stage 4.    -   Stage 5: Cells resulting from Stage 4 are treated with the        appropriate factors, including in certain embodiments: (i) one        or more of T3, T4 or an analogue thereof; (ii) an ALK5        inhibitor; or (iii) both of (i) and (ii) and cultured,        optionally and preferably at the air-liquid interface, to induce        differentiation to cells expressing markers characteristic of        pancreatic endoderm/endocrine precursor cells.    -   Stage 6: Cells resulting from Stage 5 cells are treated with the        appropriate factors including in certain embodiments: (i) one or        more of T3, T4 or an analogue thereof; (ii) an ALK5        inhibitor, (iii) one or more of an aurora kinase inhibitor, an        RSK inhibitor and an inhibitor of protein methyltransferase        DOT1L; (iv) both of (i) and (ii); (v) (i), (ii) and        (iii); (vi) (i) and (iii); or (vii) (ii) and (iii) and cultured,        optionally and preferably at the air-liquid interface, to induce        differentiation into cells expressing markers characteristic of        pancreatic endocrine cells.    -   Stage 7: Cells resulting from Stage 6 cells are treated with        appropriate factors including in certain embodiments: (i) one or        more of T3, T4 or thereof; (ii) an ALK5 inhibitor, (iii) an        anti-oxidant, (iv) one or more of an aurora kinase inhibitor, an        RSK inhibitor and an inhibitor of protein methyltransferase        DOT1L; (v) (i) and (ii); (vi) (i) and (iii); (vii) (i) and        (iv); (viii) (ii) and (iii); (ix) (ii) and (iv); (x) (i), (ii),        and (iii); (xi) (i), (iii), and (iv): (xii) (ii), (iii), and        (iv); (xiii) (i), (ii) and (iv); (xiv) (iii) and (iv); or (xv)        (i), (ii), (iii) and (iv) and cultured, optionally and        preferably at the air-liquid interface, to induce formation of        pancreatic endocrine cells that express single hormonal insulin        and are PDX1, NKX6.1 and MAFA positive and which have a higher        level of expression of MAFA than the Stage 6 cells and the        resulting cell population has a higher percentage of both MAFA        positive and single hormonal insulin expressing cells than the        Stage 6 cells.        While the invention in certain embodiments encompasses        differentiating pluripotent stem cells (e.g. pre-Stage 1 cells)        to Stage 7 cells, the invention also encompasses differentiating        cells at other stages towards Stage 7. In particular, the        invention encompasses differentiation of Stage 4 to Stage 7        cells. Moreover, although the process is described in discrete        stages, the treatment, as well as the progress of the cells        through the differentiation process, may be sequential or        continuous. Moreover, differentiation of pluripotent stem cells        to Stage 6 or Stage 7 cells can be carried out in suspension        cultures.

The efficiency of differentiation may be determined by exposing atreated cell population to an agent, such as an antibody, thatspecifically recognizes a protein marker expressed by the differentiatedcells of interest. Methods for assessing expression of protein andnucleic acid markers in cultured or isolated cells are standard in theart. These methods include RT-PCR, Northern blots, in situ hybridization(see, e.g., Current Protocols in Molecular Biology (Ausubel et al., eds.2001 supplement)), and immunoassays such as immunohistochemical analysisof sectioned material, Western blotting, and for markers that areaccessible in intact cells, flow cytometry analysis (FACS) (see, e.g.,Harlow and Lane, Using Antibodies: A Laboratory Manual, New York: ColdSpring Harbor Laboratory Press (1998)).

The differentiated cells may also be further purified. For example,after treating pluripotent stem cells with the methods of the presentinvention, the differentiated cells may be purified by exposing atreated cell population to an agent, such as an antibody, thatspecifically recognizes a protein marker characteristically expressed bythe differentiated cells being purified.

Any suitable growth medium containing sufficient quantities of vitamins,minerals, salts, glucose, amino acids and carrier proteins desirable forcells differentiation may be used for the various Stages 1 through 7.However, preferably, the following are used: Stage 1—MCDB-131 (availablefrom (Life Technologies Corporation, Grand Island, N.Y.) or RPMI(available from Sigma-Aldrich)); Stage 2—MCDB-131 or Dulbecco's ModifiedEagle's Medium F12 (“DMEM—F12”); Stage 3 through 5—MCDB-131, BLAR (Table1), or DMEM; and Stages 6 and 7—BLAR or CMRL (Life Technologies).Preferably, the glucose concentration of the medium is kept at or, morepreferably, lower than about 10 mM for Stages 1 through 4 and greaterthan about 10 mM for Stages 5 through 7.

-   -   Stage 1: Differentiation of pluripotent cells into cells        expressing markers characteristic of definitive endoderm cells.

Pluripotent stem cells may be differentiated into cells expressingmarkers characteristic of definitive endoderm cells by any method knownin the art, or by any method proposed in the invention. Methodsreportedly useful for differentiating pluripotent stem cells into cellsexpressing markers characteristic of the definitive endoderm lineage aredisclosed in: D'Amour et al., Nature Biotechnology 23, 1534-1541 (2005);Shinozaki et al., Development 131, 1651-1662 (2004); McLean et al., StemCells 25, 29-38 (2007); D'Amour et al., Nature Biotechnology 24,1392-1401 (2006). Additional suitable differentiation methods aredisclosed in: U.S. Patent App. Pub. No. 2007/0254359; U.S. Patent App.Pub. 2009/0170198; U.S. Patent App. Pub. 2011/0091971; U.S. Patent App.Pub. 2010/0015711; U.S. Patent App. Pub. 2012/0190111; U.S. Patent App.Pub. 2012/0190112; and U.S. Patent App. Pub. 2012/0196365. Thesedisclosures are incorporated herein by reference in their entireties asthey pertain to the differentiation of pluripotent stem cells intodefinitive endoderm cells.

In one embodiment, the pluripotent cells are treated with a suitablegrowth medium, preferably MCDB-131 or RPMI. The medium is preferablysupplemented with a growth differentiation factor, such as growthdifferentiation factor 8 (“GDF8”), and a glycogen synthase kinase-3 β(“GSK3β”) inhibitor, such as the cyclic aniline-pyridintriazinecompounds disclosed in U.S. Patent App. Pub. No. 2010/0015711(incorporated herein in its entirety by reference) to inducedifferentiation into cells expressing markers characteristic ofdefinitive endoderm cells. A preferred GSK3β inhibitor is14-prop-2-en-1-yl-3,5,7,14,17,23,27-heptaazatetracyclo[19.3.1.1˜2,6˜.1˜8,12˜]heptacosa-1(25),2(27),3,5,8(26),9,11,21,23-nonaen-16-one (“MCX Compound”).Treatment may involve contacting pluripotent stem cells with a mediumsupplemented with about 50 ng/ml to about 150 ng/ml, alternatively about75 ng/ml to about 125 ng/ml, preferably about 100 ng/ml of GDF8. Thetreatment may also involve contacting cells with about 0.1 to about 5μM, alternatively about 0.5 to about 2.5 μM, preferably about 1 μM ofMCX Compound. The pluripotent cells may be cultured for about two tofive days, preferably about two to three days, to facilitatedifferentiation into cells expressing markers characteristic of thedefinitive endoderm cells.

In a preferred embodiment, the cells are cultured in the presence ofGDF8 and MCX Compound for one day, followed by culturing in the presenceof GDF8 and a lower concentration of MCX Compound for one day, followedby culturing in the presence of GDF8 for one day in the absence of MCXCompound. In particular, the cells are cultured in the presence of GDF8and about 1 μM MCX Compound for one day, followed by culturing in thepresence of GDF8 and about 0.1 μM MCX Compound for one day, followed byculturing in the presence of GDF8 for one day in the absence of MCXCompound. Alternatively, the cells may be cultured in the presence ofGDF8 and about 1 μM MCX Compound for one day, followed by culturing inthe presence of GDF8 and about 0.1 μM MCX Compound for one day.

Alternatively, the pluripotent stem cells may be cultured in mediumcontaining activin A in the absence of serum, then culturing the cellswith activin A and serum, and then culturing the cells with activin Aand serum of a different concentration as disclosed in D'Amour et al.,Nature Biotechnology 23, 1534-1541 (2005). As yet another alternative,the pluripotent stem cells may be differentiated into cells expressingmarkers characteristic of definitive endoderm cells by culturing thepluripotent stem cells in medium containing activin A in the absence ofserum, then culturing the cells with activin A with serum as disclosedin D'Amour et al., Nature Biotechnology, 2005. Still further,pluripotent stem cells may be differentiated into cells expressingmarkers characteristic of the definitive endoderm lineage by culturingthe pluripotent stem cells in medium containing activin A and a WNTligand in the absence of serum, then removing the WNT ligand andculturing the cells with activin A with serum as disclosed in D'Amour etal., Nature Biotechnology 24, 1392-1401 (2006).

In one embodiment of the invention, pluripotent stem cells are treatedwith activin A and WNT3A to result in the formation of cells expressingmarkers characteristic of definitive endoderm cells. Treatment mayinvolve contacting pluripotent stem cells with about 50 ng/ml to about150 ng/ml, alternatively about 75 ng/ml to about 125 ng/ml,alternatively about 100 ng/ml of activin A. The treatment may alsoinvolve contacting the cells with about 10 ng/ml to about 50 ng/ml,alternatively about 15 ng/ml to about 30 ng/ml, alternatively about 20ng/ml of WNT3A. The pluripotent cells may be cultured for approximatelythree days to arrive at the definitive endoderm cells. In oneembodiment, the cells are cultured in the presence of activin A andWNT3A for one day followed by culturing in the presence of activin A(without WNT3A being present) for the remainder.

Formation of cells expressing markers characteristic of definitiveendoderm cells may be determined by testing for the presence of themarkers before and after following a particular protocol. Pluripotentstem cells typically do not express such markers. Thus, differentiationof pluripotent cells can be detected when cells begin to express markerscharacteristic of definitive endoderm.

-   -   Stage 2: Differentiation of cells expressing markers        characteristic of definitive endoderm cells into cells        expressing markers characteristic of gut tube cells.

The cells expressing markers characteristic of the definitive endodermcells may be further differentiated into cells expressing markerscharacteristic of gut tube cells in a growth medium, such as MCDB-131 orDMEM F12. In one embodiment, the formation of cells expressing markerscharacteristic of gut tube cells includes culturing the cells expressingmarkers characteristic of the definitive endoderm cells with a mediumcontaining fibroblast growth factor (“FGF”), preferably FGF7 or FGF10,to differentiate the cells. For example, the cell culture may includefrom about 10 ng/ml to about 75 ng/ml, alternatively from about 25 ng/mlto about 75 ng/ml, still alternatively from about 30 ng/ml to about 60ng/ml, alternatively about 50 ng/ml of a fibroblast growth factor,preferably FGF7 or FGF10, more preferably FGF7, and most preferablyabout 25 ng/ml FGF7. The cells may be cultured under these conditionsfor about two to three days, preferably about two days.

In another embodiment, the formation of cells expressing markerscharacteristic of gut tube cells includes culturing the cells expressingmarkers characteristic of the definitive endoderm lineage with afibroblast growth factor, preferably, FGF7 or FGF10, and ascorbic acid(Vitamin C). The culture medium may include from about 0.1 mM to about0.5 mM ascorbic acid, alternatively from about 0.2 mM to about 0.4 mMascorbic acid, alternatively about 0.25 mM of ascorbic acid. The cellculture may also include from about 10 ng/ml to about 35 ng/ml,alternatively from about 15 ng/ml to about 30 ng/ml, alternatively about25 ng/ml of the fibroblast growth factor, preferably FGF7 or FGF10, morepreferably FGF7. For example, the cell culture may include about 0.25 mMof ascorbic acid and about 25 ng/ml of FGF7. In one embodiment, theStage 1 cells are treated for 2 days with FGF7 and ascorbic acid.

-   -   Stage 3: Differentiation of cells expressing markers        characteristic of gut tube cells into cells expressing markers        characteristic of foregut endoderm cells.

The gut tube cells resulting from carrying out Stage 2 may be furtherdifferentiated into Stage 3 cells, or cells expressing markerscharacteristic of the foregut endoderm, by culturing these cells in agrowth medium such as MCDB-131, DMEM, or a custom media such as BLAR(Table I). The medium may be supplemented with: (i) a fibroblast growthfactor, preferably, FGF7 or FGF10 and more preferably FGF7; (ii)retinoic acid (“RA”); (iii) a Sonic Hedgehog (“SHH”) signaling pathwayantagonist (such as Smoothened Antagonist 1 (“SANT-1”) which is1-piperazinamine,N-[(3,5-dimethyl-1-phenyl-1H-pyrazol-4-yl)methylene]-4-(phenylmethyl)-or((E)-4-benxyl-N-((3,5-dimethyl-1-phenyl-1H-pyrazol-4-yl),ethylene-piperazin-1-amine),HPI-1 which is 2-methoxyethyl1,4,5,6,7,8-hexahydro-4-(3hydroxyphenyl)-7-(2-methoxyphenyl)-2-methyl-5-oxo-3-quinolinecarboxylate,and preferably SANT-1; (iv) a protein kinase C (“PKC”) activator, suchas((2S,5S)-(E,E)-8-(5-(4-(trifluoromethyl)phenyl)-2,4-pentadieneoylamino)benzolactam)(“TPB”), phorbol-12,13-dibutyrate (“PDBu”),phorbol-12-myristate-13-acetate (“PMA”) or indolactam V (“ILV”) andpreferably TPB; (v) a bone morphogenic protein (“BMP”) inhibitor, suchas LDN-193189, Noggin, or Chordin and preferably LDN-193189; and (vi)ascorbic acid. Alternatively, a Smoothened (“SMO”) receptor inhibitor(such as MRT10(N[[[3-benzoylamino)phenyl]amino]thioxomethyl]-3,4,5-trimethoxybenzamide))or cyclopamine may also be used. For example, the cell culture mayinclude from about 100 nM to about 500 nM, alternatively from about 100nM to about 400 nM, alternatively about 200 nM of a PKC activator. Thecells may be cultured in the presence of these growth factors, smallmolecule agonists and antagonists for about two to four days, preferablyabout two to three days, more preferably about two days.

Alternatively, the Stage 2 cells may be differentiated into Stage 3cells by culturing these cells in a culture medium supplemented with aSMO receptor inhibitor, SANT-1, retinoic acid, and Noggin. The cells maybe cultured for approximately two to four days, preferably about twodays.

In one embodiment, the medium is supplemented with: from about 10 ng/mlto about 35 ng/ml, alternatively from about 15 ng/ml to about 30 ng/ml,alternatively about 25 ng/ml of the fibroblast growth factor, preferablyFGF7 or FGF10, more preferably FGF7; from about 0.1 mM to about 0.5 mMascorbic acid, alternatively from about 0.2 mM to about 0.4 mM,alternatively about 0.25 mM of ascorbic acid; from about 0.1 μM to about0.4 μM of SANT-1; from about 100 to about 300 nM of TPB; and from about50 nM to about 200 nM, and about 100 nM of LDN-193189. In anotherembodiment, the medium is supplemented with about 25 ng/ml of FGF-7,about 1 μM of retinoic acid, about 0.25 μM of SANT-1, about 200 nM ofTPB, about 100 nM of LDN-193189, and about 0.25 mM of ascorbic acid.

In one embodiment, the medium is supplemented with from about 0.1 μM toabout 0.3 μM of SANT-1, from about 0.5 μM to about 3 μM of retinoic acidand from about 75 ng/ml to about 125 ng/ml of Noggin.

-   -   Stage 4 through Stage 7: Differentiation of cells expressing        markers characteristic of foregut endoderm cells into cells        expressing markers characteristic of a mature phenotype        pancreatic endocrine cells by treatment with culture medium        supplemented with one or both of a thyroid hormone and ALK        inhibitor along with one or more of an aurora kinase inhibitor,        an RSK inhibitor, and an inhibitor of protein methyltransferase        DOT1L, preferably by culturing at the air-liquid interface.

Although in one embodiment, the present invention contemplates culturingat the air-liquid interface for all stages in the path for pluripotentcell to pancreatic endocrine cell, the invention preferably provides forthe formation of Stage 1 to Stage 4 cells in planar or submerged cultureand Stage 5, 6, and 7 cells by culturing the cells at the air-liquidinterface. In other embodiments, the present invention relates to astepwise method of differentiation pluripotent cells comprisingculturing Stage 4, 5 and 6 cells at the air-liquid interface. In certainembodiments, cells cultured during Stages 4 through 7, may be culturedat the air-liquid interface. In other embodiments, only late Stage 4 toStage 6 cells, or Stage 5 and Stage 6 cells, are cultured at theair-liquid interface. In yet another alternative embodiment, Stage 1through 4 are carried out by culturing the cells in submerged planarcultures and Stage 5 through 7 are carried out by culturing in submergedsuspension cultures.

Additionally, culturing during one or more, and preferably all of,Stages 5, 6, and 7 is carried out in the presence of one or more of T3,T4 and their analogues, an ALK5 inhibitor, or both one or more of T3, T4and their analogues and an ALK5 inhibitor. In preferred embodiments,culturing during one or more, and preferably all of, Stages 5, 6, and 7is preferably carried out in the presence of T3 and an ALK5 inhibitorand more preferably in the presence of T3 and ALK5 inhibitor II.Suitable amounts of the thyroid hormones or their analogues are about 0to about 1000 nM, alternatively about 10 to about 900 nM, alternativelyabout 100 to about 800 nM, alternatively about 200 to about 700 nM,alternatively about 300 to about 600 nM, alternatively about 400 toabout 500 nM, alternatively about 1 to about 500 nM, alternatively about1 to about 100 nM, alternatively about 100 to about 1000 nM,alternatively about 500 to about 1000 nM, alternatively about 100 toabout 500 nM, alternatively about 1 μM, and preferably about 0.1 to 1μM. The amounts of ALK5 inhibitor are about 250 nM to 2 μM,alternatively about 300 to about 2000 nM, alternatively about 400 toabout 2000 nM, alternatively about 500 to about 2000 nM, alternativelyabout 600 to about 2000 nM, alternatively about 700 to about 2000 nM,alternatively about 800 to about 2000 nM, alternatively about 1000 toabout 2000 nM, alternatively about 1500 to about 2000 nM, alternativelyabout 250 to about 1000 nM, alternatively about 250 to about 500 nM,alternatively about 300 to about 1000 nM, alternatively about 400 toabout 1000 nM, alternatively about 500 to about 1000 nM, alternativelyabout 600 to about 1000 nM, alternatively about 700 to about 1000 nM,alternatively about 800 to about 1000 nM, alternatively about 500 nM,alternatively about 10 μM, and preferably about 10 μM.

When cells are cultured at the air-liquid interface (“ALI”), the cellsmay be cultured on a porous substrate such that the cells are in contactwith air on the top side and with cell culture media at the bottom side.For example, a sufficient volume of media may be added to the bottom ofa culture vessel containing the porous substrate (e.g. a filter insert)such that the media contacts the bottom surface of cells residing on thesubstrate but does not encapsulate or submerge them. Suitable poroussubstrates can be formed of any material that will not adversely affectthe growth and differentiation of the cells. Exemplary porous substratesare made of polymers such as polyethylene terephthalate (“PET”),polyester, or polycarbonate. Suitable porous substrates may be coated oruncoated. In one embodiment, the coating may be MATRIGEL™. In oneembodiment of the invention, the porous substrate is a porous filterinsert, which may be coated with MATRIGEL™. In one embodiment of theinvention, the porous substrate is an uncoated filter insert. Theporosity of the substrate should be sufficient to maintain cellviability and promote differentiation of the cells. Suitable substratesinclude filter inserts having a pore size of from about 0.3 to about 3.0μm, from about 0.3 to about 2.0 μm, about 0.3 to about 1.0 μm, fromabout 0.3 to about 0.8 μm, from about 0.3 to about 0.6 μm, from about0.3 to about 0.5 μm, from about 0.3 to about 3.0 μm, from about 0.6 toabout 3.0 μm, from about 0.8 to about 3.0 μm, from about 1.0 to about3.0 μm, from about 2.0 to about 3.0 μm, preferably about 0.4 μm and apore density of from about 50 to about 120 million pores/cm², from about60 to about 110 million pores/cm², from about 70 to about 100 millionpores/cm², preferably from about 80 to about 100 million pores/cm², fromabout 90 to about 100 million pores/cm², and more preferably about 100million pores/cm².

The media may be exchanged or refreshed every other day or, preferably,daily. The cells grown on top of the porous substrate are generally notsingle cells, but rather they are in the form of a sheet or exist as anaggregate cell cluster. Cells cultured at the ALI may experience higheroxygen tension as compared to cells submerged in media.

The present invention encompasses formation of Stage 4 to 7, preferablyStage 5 to 7, cells at the air-liquid interface. The cells may be formedby differentiating pluripotent stem cells or by further differentiatingStage 3, 4, 5, or 6 cells. Stage 4 cells may be cultured entirely at theair-liquid interface or the cells may be cultured in submerged planarculture during the early portion of Stage 4, meaning about one to twodays and then cultured at the air-liquid interface for the latterportion of Stage 4, meaning about day two to day three. Preferably,Stage 4 is not carried out at the ALI, but rather in submerged culture.

In one embodiment, the present invention provides a method for producingcells expressing markers characteristic of pancreatic endocrine cellsfrom pluripotent stem cells, comprising culturing pluripotent stemcells, differentiating the pluripotent stem cells into cells expressingmarkers characteristic of the foregut endoderm; differentiating thecells expressing markers characteristic of the foregut endoderm intocells expressing markers characteristic of the pancreatic endocrinecellsby culturing, optionally, at the air-liquid interface. The method mayinclude treatment with a medium supplemented with one or both of (i) T3,T4 or their analogues, (ii) an ALK5 inhibitor, or both (i) and (ii). Themethod may include differentiating the cells expressing markerscharacteristic of foregut endoderm cells (Stage 3 cells) into cellsexpressing markers characteristic of pancreatic foregut precursor cells(Stage 4 cells) by treatment with a medium supplemented with (i) one orboth of T3, T4 or their analogues, (ii) ALK5 inhibitor or both (i) and(ii) and culturing in a planar culture. The method may also includedifferentiating cells expressing markers characteristic of pancreaticforegut precursor cells (Stage 4 cells) into cells expressing markerscharacteristic of the pancreatic endocrine cells (Stage 6 cells) bytreatment with a medium supplemented with (i) one or both of T3, T4 ortheir analogues, (ii) ALK5 inhibitor or both (i) and (ii) and culturingin a planar culture or, and preferably, culturing at the air-liquidinterface. The method further includes differentiating Stage 6 cellsinto cells expressing markers characteristic of pancreatic endocrinecells and that have a more mature phenotype as compared to Stage 6(Stage 7 cells) by treatment with a medium supplemented with (i) one orboth of T3, T4 or their analogues, (ii) ALK5 inhibitor or both (i) and(ii) along with an one or more of an aurora kinase inhibitor, an RSKinhibitor, and an inhibitor of protein methyltransferase DOT1L and,optionally but preferably an anti-oxidant such as Vitamin E or,preferably, acetyl cysteine. The amount of acetyl cysteine that isuseful is about 0.1 to about 2 mM. The amount of Vitamin E is about 0.1to about 10 μM. In yet another embodiment, the method further includescarrying out Stage 6 by treatment of Stage 5 cells with a mediumsupplemented with (i) one or both of T3, T4 or their analogues, (ii)ALK5 inhibitor or both (i) and (ii) along with one or more of an aurorakinase inhibitor, an RSK inhibitor, and an inhibitor of proteinmethyltransferase DOT1L. In still another embodiment, Stage 6 is carriedout by treatment of Stage 5 cells with a medium supplemented with (i)one or both of T3, T4 or their analogues, (ii) ALK5 inhibitor or both(i) and (ii) along with one or more of an aurora kinase inhibitor, anRSK inhibitor, and an inhibitor of protein methyltransferase DOT1Lfollowed by carrying out Stage 7 by treatment with a medium supplementedwith (i) one or both of T3, T4 or their analogues, (ii) ALK5 inhibitoror both (i) and (ii) along with an one or more of an aurora kinaseinhibitor, an RSK inhibitor, and an inhibitor of proteinmethyltransferase DOT1L and, optionally but preferably an anti-oxidantsuch as Vitamin E or, preferably, acetyl cysteine.

One embodiment of the invention is a method of forming pancreaticendocrine cells expressing markers characteristic of maturing beta cells(Stage 7 cells) comprising differentiating cells expressing markerscharacteristic of the pancreatic foregut precursor cells (Stage 4 cells)into cells expressing markers characteristic of Stage 7 cells byculturing, preferably at the air-liquid interface. In anotherembodiment, the methods of the invention result in the formation ofStage 6 cells, or cells that are immature beta cells, The method,preferably at least during Stages 5 through 7, includes treatment with amedium supplemented with T3, T4, or an analogue thereof, an ALK5inhibitor, or both.

Culturing of the cells at the air-liquid interface includes seeding thecells on a porous substrate such as a porous filter insert. In certainembodiments, the substrate pore size may range from about 0.3 to about 3microns. Seeding may be accomplished by releasing cells as single cellsfrom monolayer cultures or clusters of cells from monolayer culturesinto a suspension and subsequently aliquoting the single cell suspensionor suspended cell culture onto a porous substrate at the ALI. The cellsmay be seeded onto the porous substrate from a suspension having about1000 cells/μl to about 100,000 cells/μl, about 1000 cells/μl to about90,000 cells/μl, about 1000 cells/μl to about 80,000 cells/μl, about1000 cells/μl to about 70,000 cells/μl, about 1000 cells/μl to about60,000 cells/μl, about 1000 cells/μl to about 50,000 cells/μl, about1000 cells/μl to about 40,000 cells/μl, about 1000 cells/μl to about30,000 cells/μl, about 1000 cells/μl to about 20,000 cells/μl, about1000 cells/μl to about 10,000 cells/μl, about 1000 cells/μl to about5000 cells/μl, about 5000 cells/μl to about 100,000 cells/μl, about10,000 cells/μl to about 100,000 cells/μl, about 20,000 cells/μl toabout 100,000 cells/μl, about 30,000 cells/μl to about 100,000 cells/μl,about 40,000 cells/μl to about 100,000 cells/μl, about 50,000 cells/μlto about 100,000 cells/μl, about 60,000 cells/μl to about 100,000cells/μl, about 20,000 cells/μl to about 80,000 cells/μl, about 30,000cells/μl to about 70,000 cells/μl, about 40,000 cells/μl to about 60,000cells/μl, and preferably about 50,000 cells/μl. The cells may be seededas droplets of the cell suspension containing individual cells oraggregates or clusters of cells. The resulting cell deposit may containabout 5×10⁶ to about 5×10⁷ cells/cm², about 6×10⁶ to about 5×10⁷cells/cm², about 7×10⁶ to about 5×10⁷ cells/cm², about 8×10⁶ to about5×10⁷ cells/cm², about 9×10⁶ to about 5×10⁷ cells/cm², about 1×10⁷ toabout 5×10⁷ cells/cm², about 2×10⁷ to about 5×10⁷ cells/cm², about 3×10⁷to about 5×10⁷ cells/cm², about 4×10⁷ to about 5×10⁷ cells/cm², about5×10⁶ to about 4×10⁷ cells/cm², about 5×10⁶ to about 3×10⁷ cells/cm²,about 5×10⁶ to about 2×10⁷ cells/cm², about 5×10⁶ to about 1×10⁷cells/cm², about 5×10⁶ to about 9×10⁶ cells/cm², about 5×10⁶ to about8×10⁶ cells/cm², about 5×10⁶ to about 7×10⁶ cells/cm², about 5×10⁶ toabout 6×10⁶ cells/cm², about 7×10⁶ to about 4×10⁷ cells/cm², about 8×10⁶to about 3×10⁷ cells/cm², about 9×10⁶ to about 2×10⁷ cells/cm², andpreferably on the order of about 1×10⁷ cells/cm².

In another embodiment, the invention refers to a method of enhancing thenumber of single hormone positive cells (e.g. cells that co-expressNKX6.1 and insulin or cells that co-express NKX6.1 and chromogranin byculturing and differentiating a population of PDX1 and NKX6.1co-expressing cells, preferably at an air-liquid interface. In anotherembodiment, pancreatic endoderm cells cultured at the air-liquidinterface are further differentiated to pancreatic endocrine cells bytreatment with a compound selected from the following: ALK5 inhibitor,BMP inhibitor, gamma-secretase inhibitor, Ephrin ligands, EphBinhibitor, PKC inhibitor, EGFr inhibitor, retinoic acid, vitamin C,T3/T4, glucose, cell cycle regulators, WNT regulators, SHH inhibitor,aurora inhibitor, anti-oxidants, vitamin E, acetyl-cysteine, orcombinations thereof.

In further embodiments, the present invention relates to a stepwisemethod of differentiating pluripotent cells that includes culturingStage 4 through Stage 6 cells in a media containing sufficient amountsof (i) one or more of T3, T4 and their analogues, (ii) an ALK5inhibitor, or both (i) and (ii) and further culturing the Stage 6 cellsin a media that optionally, and preferably, contains one or more of anaurora kinase inhibitor, an RSK inhibitor, and an inhibitor of proteinmethyltransferase DOT1L, and an antioxidant to generate pancreaticendocrine cells and populations of pancreatic endocrine cells thatexpress insulin, PDX1, NKX6.1, and MAFA.

In some embodiments, at least 10% of the cells of the resulting cellpopulation express insulin, PDX1, NKX6.1, and MAFA. In otherembodiments, at least 20% of the cells of the population expressinsulin, PDX1, NKX6.1, and MAFA. In other embodiments, at least 30% ofthe cells of the population express insulin, PDX1, NKX6.1, and MAFA. Instill other embodiments, at least 40% of the cells of the populationexpress insulin, PDX1, NKX6.1, and MAFA. In yet other embodiments, atleast 50% of the cells of the population express insulin, PDX1, NKX6.1,and MAFA. In alternative embodiments, at least 60% of the cells expressinsulin, PDX1, NKX6.1, and MAFA. In still other alternative embodiments,at least 70% of the cells of the population express insulin, PDX1,NKX6.1, and MAFA. In yet other embodiments, at least 80% of the cells ofthe population express insulin, PDX1, NKX6.1, and MAFA. In otherembodiments, at least 90% of the cells of the population expressinsulin, PDX1, NKX6.1, and MAFA. In alternative embodiments, at least91, 92, 93, 94, 95, 96, 97, 98, or 99% of the cells of the populationexpress insulin, PDX1, NKX6.1, and MAFA.

-   -   Stage 4: Differentiation of cells expressing markers        characteristic of foregut endoderm cells into cells expressing        markers characteristic of pancreatic foregut precursor cells.

In one embodiment, the methods of the invention include treating Stage 3cells with a differentiation medium that may be any suitable growthmedium and preferably is MCDB-131, DMEM, or a custom media such as BLAR(Table I). The medium may be supplemented with one or more of thefollowing: (a) an ALK5 inhibitor selected from the group consisting of:TGF-β receptor inh V, TGF-β receptor inh I, TGF-β receptor inh IV, TGF-βreceptor inh VII, TGF-β receptor inh VIII, TGF-β receptor inh II, TGF-βreceptor inh VI, TGF-β receptor inh III, TGF-β inhibitor SB431542,SD-208, ITD-1, LY2109761, A83-01, LY2157299, ALK5i and ALK5 inhibitorII; (b) a thyroid hormone selected from the group consisting of T3, T4,analogues of T3, analogues of T4 and mixtures thereof (c) a SHHsignaling pathway antagonist selected from SANT-1 or HIP-1; (d) a BMPreceptor inhibitor selected from LDN-193189, Noggin or Chordin; (e) aPKC activator selected from TPB, PPBu, PMA, and ILV; (f) a fibroblastgrowth factor selected from FGF-7 or FGF-10; (g) retinoic acid; and (h)ascorbic acid. For example, a growth medium such as MCDB131 or, andpreferably, BLAR may be supplemented with a SHH signaling pathwayantagonist (such as SANT-1 or HPI-1), a BMP inhibitor (such asLDN-193189, Noggin or Chordin), ascorbic acid, and a PKC activator (suchas TPB, PDBu. PMA or ILV), to provide a useful differentiation media.Culturing Stage 3 cells in such medium for about two to four days,preferably about two to three days, more preferably about three daysusually is sufficient to differentiate the Stage 3 cells into Stage 4cells. In another embodiment, the medium may be supplemented with a SMOinhibitor and SHH signaling pathway antagonist. In a preferredembodiment, the Stage 3 cells may be treated with a medium supplementedwith about 0.25 μM SANT-1; about 100 nM RA; about 2 ng/ml FGF7; about100 nM LDN-193189; and about 0.25 mM ascorbic acid; and about 200 nM forthree days.

In Stage 4, cells may be cultured at the air-liquid interface, eitherduring the entire stage or, and preferably, after about 2 to 3 days ofplanar culturing. Specifically, the present invention provides an invitro cell culture for differentiating cells derived from pluripotentstem cells at the air-liquid interface comprising: (a) a culture vessel;(b) a volume of growth medium within said vessel sufficient to fill onlya portion of the volume of the vessel; (c) air within the vessel thatfills a portion of the vessel adjoining the medium; (d) a poroussubstrate located at the interface between the medium and the air; and(e) cells derived from pluripotent stem cells disposed upon the surfaceof the substrate such that the medium contacts only a portion of thesurface of the cells. Alternatively, Stage 4 may be carried out entirelyin planar culture.

In a further embodiment, the cells at the completion of Stage 4 may betreated with a Rho-associated kinase (“ROCK”) inhibitor such as Y27632((1R,4r)-4-((R)-1-aminoethyl)-N-(pyridin-4-yl)cyclohexanecarboxamide),GSK269962(N-[3-[[2-(4-Amino-1,2,5-oxadiazol-3?-yl)-1-ethyl-1H-imidazo[4,5-c]pyridin-6-yl]oxy]phenyl]-4-[2-(4-morpholinyl)ethoxy]benzamide),H1152((S)-(+)-2-Methyl-1-[(4-methyl-5-isoquinolinyl)sulfonyl]homopiperazine,2HCl) and, SR3677(N-[2-[2-(Dimethylamino)ethoxy]-4-(1H-pyrazol-4-yl)phenyl-2,3-dihydro-1,4-benzodioxin-2-carboxamidedihydrochloride). In certain embodiments about 1 to 20 μM, alternativelyabout 1 to 15 μM, alternatively about 1 to 10 μM, preferably about 10 μMof the ROCK inhibitor may be used.

In certain embodiments, only late Stage 4 cells, meaning cells that havebeen cultured for 1 to 2 days in planar cultures, may subsequently becultured at the air-liquid interface for completion of Stage 4. In oneembodiment, only late Stage 4 cells that were treated with a ROCKinhibitor are cultured at the air-liquid interface. In otherembodiments, 0.5 to about 0.75×10⁵ cells/micro liter are seeded to becultured at the air-liquid interface; alternatively, about 2 to 6×10⁶cells are seeded to be cultured at the air-liquid interface. In certainembodiments, the cells may be treated with a cell detachment solution,such as a solution containing proteolytic and collagenolytic enzymessuch as TrypLE™, Accutase™, or Dispase™ prior to culturing at theair-liquid interface.

In an alternate embodiment, Stage 3 cells may be treated with adifferentiation medium comprising a growth medium supplemented with anALK5 inhibitor, Noggin, and a PKC activator, such as TPB. In certainembodiments, the medium may be supplemented with about 0.1 μM ALK5inhibitor, about 100 ng/mL of Noggin, and about 500 nM TPB. The cellculture may be in a monolayer format. The treatment may last for a totalof about three days. In certain embodiments, the cells may be treatedfor two days and then on the last day the cells may be treated withproteolytic enzymes, collagenolytic enzymes or both, such as Dispase™,and broken into cell clusters having a diameter of less than about 100microns followed by culturing in the presence of an ALK5 inhibitor andLDN-193189. In certain embodiments, the cell clusters having a diameterof less than about 100 microns may be cultured in a medium supplementedwith about 200 nM ALK5 inhibitor and about 100 nM LDN-193189. In analternate embodiment, culturing Stage 4 cells at the air-liquidinterface may significantly enhance pancreatic endoderm markers alongwith endocrine-related markers. Accordingly, the invention provides formethods of enhancing pancreatic endoderm and endocrine-related markersby culturing Stage 4 cells at the air-liquid interface

-   -   Stage 5: Differentiation of cells expressing markers        characteristic of pancreatic foregut precursor cells into cells        expressing markers characteristic of pancreatic        endoderm/endocrine precursor cells.

In one embodiment, the methods of the invention include treating Stage 4cells with a differentiation medium that may be any suitable growthmedium and preferably is MCDB-131, DMEM or, and preferably, a custommedia such as BLAR (Table I). The medium may be supplemented with one ormore of the following: (a) an ALK5 inhibitor selected from the groupconsisting of: TGF-β receptor inh V, TGF-β receptor inh I, TGF-βreceptor inh IV, TGF-β receptor inh VII, TGF-β receptor inh VIII, TGF-βreceptor inh II, TGF-β receptor inh VI, TGF-β receptor inh III, TGF-βinhibitor SB431542, SD-208, ITD-1, LY2109761, A83-01, LY2157299, ALK5iand ALK5 inhibitor II; (b) a thyroid hormone selected from the groupconsisting of T3. T4, analogues of T3, analogues of T4 and mixturesthereof; (c) a SHH signaling pathway antagonist selected from SANT-1 orHIP-1; (d) a BMP Receptor Inhibitor selected from LDN-193189, Noggin orChordin; (e) retinoic acid; (f) ascorbic acid; (g) heparin; and (h) zincsulfate, and culturing the cells, preferably at the air-liquidinterface, for about two to four days, preferably about three days, todifferentiate the cells into Stage 5 cells. In another embodiment, thegrowth medium is also supplemented with one or both of a SMO inhibitor(such as MRT10 or cyclopamine) and a fibroblast growth factor preferablyselected from FGF-7 or FGF-10. The treatment of the Stage 4 cells iscarried out for about two to four days, preferably about 3 days todifferentiate the cells into Stage 5 cells.

In a preferred embodiment, the Stage 4 cells are differentiated intoStage 5 cells by treating the cells with a medium supplemented with fromabout 0.1 μM to about 0.4 μM of SANT-1 and preferably about 0.25 μMSANT-1, about 50 nM RA, from about 0.1 mM to about 0.5 mM ascorbic acid,alternatively from about 0.2 mM to about 0.4 mM and preferably about0.25 mM ascorbic acid, from about 50 nM to about 200 nM and preferablyabout 100 nM LDN-193189, about 1 μM of T3, and about 10000 nM ALK5inhibitor, more preferably ALK 5 inhibitor II. In still anotherembodiment, the cells are optionally and preferably also treated withabout 1 to 15 μM, alternatively about 1 to 10 μM ZnSO₄, alternativelyabout 5 to 10 μM, preferably about 10 μM about 10 μM zinc sulfate andabout 1 to 100 μg/ml, preferably about 10 μg/ml of heparin. Thetreatment of the Stage 4 cells is carried out for about two to fourdays, preferably about 3 days to differentiate the cells into Stage 5cells.

In yet another embodiment, the methods of the invention include treatingStage 4 cells with a medium supplemented with heparin, a SMO inhibitoror SHH signaling pathway antagonist, RA, a BMP receptor inhibitor and anALK5 inhibitor and culturing the cells at the air-liquid interface forabout 3 days to differentiate the cells into Stage 5 cells. In analternative embodiment, the medium may be supplemented with both a SMOinhibitor and an SHH signaling pathway antagonist, along with RA, a BMPreceptor inhibitor and an ALK5 inhibitor. Thus, in one embodiment, theStage 4 cells may be differentiated into Stage 5 cells by treating theStage 4 cells with a medium supplemented with heparin, ZnSO₄, a SMOinhibitor or SHH signaling pathway antagonist. RA, LDN-193189 and ALK5inhibitor II. In an alternative embodiment, the medium may besupplemented with both a SMO inhibitor and SHH signaling pathwayantagonist. In one embodiment, the Stage 4 cells are differentiated intoStage 5 cells by treating the cells with a medium supplemented withabout 10 μg/ml of heparin, about 0.25 μM SANT-1, about 50 nM RA, about50 nM LDN-193189, about 10 nM of T3 and about 1000 nM ALK5 inhibitor.Suitable ALK5 inhibitors include but are not limited to SD-208, ALK5inhibitor II, TGF-β receptor inh V, TGF-β receptor inh I, TGF-β receptorinh IV, TGF-1 receptor inh VII, TGF-β receptor inh VIII, TGF-β receptorinh II, TGF-β receptor inh VI, TGF-β 3 receptor inh III and combinationsthereof. The treatment of the Stage 4 cells is carried out for about twoto four days, preferably about 3 days to differentiate the cells intoStage 5 cells.

In a preferred embodiment, the ALK5 inhibitor is ALK5 inhibitor II. Inanother preferred embodiment, about 10000 nM of ALK5 inhibitor II isused. In an alternate preferred embodiment, the Stage 4 cells aretreated with a medium supplemented with about 10 μg/ml of heparin, about0.25 μM SANT-1, about 50 nM RA, about 100 nM LDN-193189, and about 10000nM of ALK5 inhibitor II. In yet another alternate embodiment, themethods of the invention include treating Stage 4 cells with a mediumsupplemented with a SMO inhibitor or SHH signaling pathway antagonist,RA, and an ALK5 inhibitor and culturing the cells, preferably at theair-liquid interface for about two days to four days, preferably about 3days, differentiate the cells into Stage 5 cells. In an alternateembodiment, the medium may be supplemented with both a SMO inhibitor andSHH signaling pathway antagonist. In one embodiment, the Stage 4 cellsare differentiated into Stage 5 cells by treating the cells with amedium supplemented with about 0.25 μM SANT-1, about 50 nM RA, about 50nM LDN-193189, about 1 μM T3 and about 1000 nM of an ALK5 inhibitor.

The amount of cells seeded for culturing at the air-liquid interface mayvary. For example, to culture the cells at the air-liquid interface,droplets of a cell suspension containing about 0.5 to 6×10⁵ cells/μl maybe seeded on a porous substrate (e.g. filter). The suspension maycontain from about 2×10⁵ cells/μl to about 6×10⁵ cells/μl; about 4×10⁷cells/μl to about 6×10⁵ cells/μl; about 5×10⁵ cells/μl to about 6×10⁵cells/μl; about 5×10⁵ cells/μl to about 6×10⁷ cells/μl; about 2×10⁵cells/μl to about 5×10⁵ cells/μl; about 2×10⁵ cells/μl to about 4×10⁷cells/μl; or about 3×10⁵ cells/μl that may be seeded onto a poroussubstrate such as a filter located at the air-liquid interface. In someembodiments, droplets of a cell suspension containing from about 0.5×10⁵cells/μl to about 0.75×10⁵ cells/μl; about 0.6×10⁷ cells/μl to about0.75×10⁵ cells/μl; or about 0.5×10⁵ cells/μl to about 0.6×10⁵ cells/μlare seeded onto a porous support to be cultured at the ALI.

In another embodiment, the methods of the invention include treatingStage 4 cells with a medium supplemented with a BMP receptor inhibitor(e.g. LDN-193189, Noggin or Chordin) and an ALK5 inhibitor for about 1day to differentiate Stage 4 cells into Stage 5 cells. For example, themedium may be supplemented with about 100 nM of LDN-193189 and withabout 100 nM of ALK5 inhibitor and about 1 μM T3. The cells may be inthe form of clusters. In certain embodiments, the cells may be treatedwith a cell detachment solution, such as a solution containingproteolytic and collagenolytic enzymes prior to culturing at theair-liquid interface.

In accordance with the foregoing method, the invention further providesa cell culture for differentiating cells expressing markerscharacteristic of the pancreatic foregut precursor into cells expressingmarkers characteristic of pancreatic endoderm/pancreatic endocrineprecursor cells comprising: (a) a culture vessel; (b) a volume of growthmedium within said vessel sufficient to fill only a portion of thevolume of said vessel; (c) air within said vessel that fills a portionof said vessel adjoining said medium; (d) a porous substrate located atthe interface between said medium and said air; and (e) cells expressingmarkers characteristic of pancreatic foregut precursor cells derivedfrom pluripotent stem cells disposed upon the surface of said substratesuch that said medium contacts only a portion of the surface of saidcells.

-   -   Stage 6: Differentiation of cells expressing markers        characteristic of pancreatic endoderm/endocrine precursor cells        into cells expressing markers characteristic of pancreatic        endocrine cells.

In one embodiment, the methods of the invention include treating Stage 5cells with a differentiation medium that may be any suitable growthmedium, preferably such as MCDB-131 or CMRL, and more preferably, acustom media such as BLAR (Table I). The medium may be supplemented withone or more of the following: (a) an ALK5 inhibitor selected from thegroup consisting of: TGF-β receptor inh V, TGF-β receptor inh I, TGF-βreceptor inh IV, TGF-β receptor inh VII, TGF-β receptor inh VIII. TGF-βreceptor inh II, TGF-β receptor inh VI, TGF-β receptor inh III, TGF-βinhibitor SB431542, SD-208, ITD-1, LY2109761, A83-01, LY2157299, ALK5iand ALK5 inhibitor II; (b) a thyroid hormone selected from the groupconsisting of T3. T4, analogues of thereof and mixtures thereof; (c) aBMP receptor inhibitor preferably selected from LDN-193189, Noggin orChordin; (d) a gamma secretase inhibitor such gamma secretase inhibitorXX, gamma secretase inhibitor XXI, gamma secretase inhibitor XVI, orDAPT; (e) ascorbic acid; (f) heparin; and (g) zinc sulfate andculturing, preferably at the air-liquid interface, for about two tofour, preferably for about three days, to differentiate the Stage 5cells into Stage 6 cells. Optionally, the medium can additionally besupplemented with one or more of an SHH signaling pathway antagonist, asmoothened receptor inhibitor, a fibroblast growth factor, and retinoicacid.

In a preferred embodiment, the Stage 5 cells may be differentiated intoStage 6 cells by treatment with a medium supplemented with about 50 nMRA, about 0.25 mM ascorbic acid, about 100 nM LDN-193189, about 10000 nMof ALK5 inhibitor and preferably ALK 5 inhibitor II, 1 μM T3, about 100nM of a gamma secretase inhibitor for about seven days. Alternatively,Stage 5 cells may be differentiated into Stage 6 cells by treatment witha medium supplemented with about 0.25 μM SANT-1, about 50 nM RA, about0.25 mM ascorbic acid, about 1000 nM ALK5 inhibitor and 1 μM T3 forabout three days. The cells may be cultured in such media for anadditional two days, or more, if desired.

Alternatively, Stage 5 cells may be differentiated into Stage 6 cells bytreatment with a medium supplemented with heparin, a SMO inhibitor orSHH signaling pathway antagonist, a BMP inhibitor, T3, T4, analoguesthereof and mixtures thereof and an ALK5 inhibitor and culturing,preferably at the air-liquid interface, for about one to seven days,alternatively about six days, alternatively about seven days. In analternate embodiment, the medium may be supplemented with both a SMOinhibitor and SHH signaling pathway antagonist. For example, the cellsmay be cultured in the medium supplemented with about 10 μg/ml ofheparin, about 0.25 μM SANT-1, about 100 nM LDN-193189, about 1000 nM ofT3 and about 500 to about 10,000 nM, alternatively about 500 nM,alternatively about 1000 mM, and alternatively about 10,000 nM of anALK5 inhibitor. Suitable ALK5 inhibitors include but are not limited toSD-208. ALK5 inhibitor II, TGF-β receptor inh V, TGF-β receptor inh I,TGF-β receptor inh IV, TGF-β receptor inh VII, TGF-β receptor inh VIII,TGF-β receptor inh II, TGF-β receptor inh VI, TGF-β receptor inh III andcombinations thereof.

In a preferred embodiment, the ALK5 inhibitor is ALK5 inhibitor II. In amore preferred embodiment, about 10000 nM of ALK5 inhibitor II is used.Accordingly, in one embodiment, Stage 5 cells may be differentiated intoStage 6 cells by treatment with a medium supplemented with heparin, SMOinhibitor or SHH signaling pathway antagonist, a BMP inhibitor, T3, T4,analogues thereof and mixtures thereof, and ALK5 inhibitor andculturing, preferably at the air-liquid interface, preferably for aboutseven days. In an alternate embodiment, the medium may be supplementedwith both an SMO inhibitor and SHH signaling pathway antagonist. Incertain embodiments, the cells may be treated with a cell detachmentsolution, such as a solution containing proteolytic and collagenolyticenzymes prior to culturing at the air-liquid interface.

In another embodiment, Stage 5 cells may be differentiated into Stage 6cells by treatment with a medium supplemented with heparin, a SMOinhibitor or SHH signaling pathway antagonist, a BMP inhibitor, T3, andALK5 inhibitor II and culturing at the air-liquid interface for about 5days to about 7 days, alternatively about 5 days, alternatively about 6days, alternatively about 7 days In these embodiments, the medium may besupplemented with about 10 μg/ml of heparin, about 0.25 μM SANT-1, about100 nM LDN-193189, about 1000 nM of T3 and about 10,000 nM of ALK5inhibitor II. In certain embodiments, the medium may be furthersupplemented with Zinc sulfate (ZnSO₄). For example, the medium may befurther supplemented with about 10 mM ZnSO₄. In an alternate embodiment,the medium may be supplemented with both a SMO inhibitor and SHHsignaling pathway antagonist

In a particularly preferred embodiment of the invention, one or more ofan aurora kinase inhibitor, preferably aurora kinase inhibitor II, anRSK inhibitor, preferably RSK inhibitor II and an inhibitor of proteinmethyltransferase DOT1L, preferably EPZ 5676, is added to the medium.The amount added may be from about 100 to 5000 nM, alternatively about1000 to 5000 nM, alternatively about 2000 to 5000 nM, alternativelyabout 3000 to 5000 nM, and preferably about 1000 to 2000 nM for theaurora kinase and RSK inhibitors and about 100 to 1000 nM for the DOT1Linhibitor, and more preferably about 1 μM to about 10 nM.

In accordance with the foregoing method, the invention further providesa cell culture for differentiating cells expressing markerscharacteristic of pancreatic endoderm/endocrine precursor cells intocells expressing markers characteristic of pancreatic endocrine cells,comprising: (a) a culture vessel; (b) a volume of growth medium withinsaid vessel sufficient to fill only a portion of the volume of saidvessel; (c) air within said vessel that fills a portion of said vesseladjoining said medium; (d) a porous substrate located at the interfacebetween said medium and said air; and (d) cells expressing markerscharacteristic of pancreatic endoderm/endocrine precursor cells derivedfrom pluripotent stem cells disposed upon the surface of said substratesuch that said medium contacts only a portion of the surface of saidcells.

In one embodiment, Stage 5 cells cultured according to embodiments ofthe invention are utilized and differentiated into Stage 6 cells, whilein other embodiments Stage 5 cells cultured according to other protocolsmay be utilized.

In another embodiment, the methods of the invention result in theformation of Stage 6 cells that are single-hormone positive. Thus, inone embodiment, the methods of the invention result in Stage 6 cells,which co-express NKX6.1, insulin, chromogranin and PDX1. In anotherembodiment, the methods of the invention result in Stage 6 cells, whichco-express NKX6.1 and insulin. In certain embodiments of the invention,the method employs BLAR a custom medium (see Table I) at Stages 4 to 6or late Stage 4 to 6, or Stages 5 and 6. The medium may be, andpreferably is, exchanged every day or alternatively every other day.

In another embodiment, the invention relates to a method of formingStage 6 cells co-expressing NKX6.1 and chromogranin comprising culturingStage 4, preferably late Stage 4 cells to Stage 6 cells at theair-liquid interface. In yet another embodiment, the invention relatesto a method of forming single hormone insulin positive cells expressingNKX6.1 Stage 6 cells by culturing Stage 4, preferably late Stage 4cells, to Stage 6 cells at the air-liquid interface.

-   -   Stage 7: Differentiation of cells expressing markers        characteristic of pancreatic endocrine cells to cells expressing        markers characteristic of pancreatic endocrine cells having a        more mature phenotype.

In one embodiment, the methods of the invention include treating Stage 6cells with a differentiation medium that may be any suitable growthmedium, preferably such as MCDB-131 or CMRL or, and more preferably, acustom media such as BLAR (Table I) The medium is supplemented with oneor more of the following: (a) an ALK5 inhibitor selected from the groupconsisting of: TGF-β receptor inh V, TGF-β receptor inh I, TGF-βreceptor inh IV, TGF-β receptor inh VII, TGF-β receptor inh VIII. TGF-βreceptor inh II, TGF-β receptor inh VI, TGF-β receptor inh III, TGF-βinhibitor SB431542, SD-208, ITD-1, LY2109761, A83-01, LY2157299, ALK5iand ALK5 inhibitor II; (b) a thyroid hormone selected from the groupconsisting of T3, T4, analogues thereof and mixtures thereof; (c)heparin; (d) zinc sulfate; (e) an antioxidant selected from the groupconsisting of vitamin E, vitamin C, acetyl cysteine, AntioxidantSupplement A1345, glutathione, peroxide dismutase, catalase, andcombinations thereof; and (f) one or more of an aurora kinase inhibitorthat is preferably aurora kinase inhibitor II, an RSK inhibitor that ispreferably RSK inhibitor II, and an inhibitor of proteinmethyltransferase DOT1L that is preferably EPZ 5676 and culturing,preferably at the air-liquid interface, for about seven to twenty-onedays, preferably for about seven to ten days, more preferably aboutseven days to differentiate the Stage 6 cells into Stage 7 Cells. In oneembodiment, the growth medium is supplemented with T3, T4, analoguesthereof and mixtures thereof, an ALK5 inhibitor, an antioxidant and anaurora kinase inhibitor, preferably aurora kinase inhibitor II or an RSKinhibitor, preferably RSK inhibitor II. The Stage 6 cells may bedifferentiated into Stage 7 cells by treatment with a mediumsupplemented with about 10000 nM of ALK5 inhibitor II, about 1000 nM ofT3, about 10 μM of 6-hydroxy-2,5,7,8-tetramethyl chroman-2-carboxylicacid (“Trolox”), and about 1 μM to about 1 μM of aurora kinase inhibitorII or RSK inhibitor II for about seven days.

In one embodiment, the Stage 6 cells may be differentiated into Stage 7cells by treatment with a medium supplemented with about 10000 nM ofALK5 inhibitor, about 1 μM of T3, about 2 μM of one or more of aurorakinase inhibitor II, RSK inhibitor II, and a EPZ 5676, and about 1 mMN-acetyl cysteine. Alternatively, one or more of about 0.25 μM SANT-1,about 50 nM RA, about 0.25 mM ascorbic acid, and about 100 nM LDN-193189also may be added.

Alternatively, Stage 6 cells may be differentiated into Stage 7 cells bytreatment with a medium supplemented with heparin, T3, T4, analoguesthereof or mixtures thereof, an ALK5 inhibitor, an antioxidant, andaurora kinase inhibitor, an RSK inhibitor, a protein methyl transferaseinhibitor of DOT1L or mixtures thereof and culturing, preferably at theair-liquid interface, for about seven to twenty-one days, alternativelyabout seven to ten days, preferably about seven days. In an alternateembodiment, the medium may be supplemented with one or more of retinoicacid, an SMO inhibitor, an SHH signaling pathway antagonist, a BMPreceptor inhibitor, and N-acetyl cysteine.

In a preferred embodiment, the ALK5 inhibitor is ALK5 inhibitor II. In amore preferred embodiment, about 10000 nM of ALK5 inhibitor II is used.Accordingly, in one embodiment, Stage 6 cells may be differentiated intoStage 7 cells by treatment with a medium supplemented with heparin, T3,T4, analogues thereof and mixtures thereof, and an ALK5 inhibitor, anantioxidant, and an aurora kinase inhibitor, an RSK inhibitor or aninhibitor of protein methyltransferase DOT1L and culturing, preferablyat the air-liquid interface, for about seven days. In certainembodiments, the cells may be treated with a cell detachment solution,such as a solution containing proteolytic and collagenolytic enzymesprior to culturing at the air-liquid interface. In a particularlypreferred embodiment of the invention, one or more of an aurora kinaseinhibitor, preferably aurora kinase inhibitor II, an RSK inhibitor,preferably RSK inhibitor II, and an inhibitor of proteinmethyltransferase DOT1L, preferably EPZ 5676 are added to the medium.The amount added may be from about 100 to 5000 nM, alternatively about1000 to 5000 nM, alternatively about 2000 to 5000 nM, alternativelyabout 3000 to 5000 nM, and preferably about 1000 nM to 2000 nM aurorakinase inhibitor, more preferably about 2000 nM aurora kinase or RSKinhibitor or about 100 to 500 nM DOT1L inhibitor, and more preferablyabout 1 μM to about 10 nM of DOT1L inhibitor.

In accordance with the foregoing method, the invention further providesa cell culture for differentiating cells expressing markerscharacteristic of pancreatic endoderm/endocrine precursor cells intocells expressing markers characteristic of mature pancreatic endocrinecells, comprising: (a) a culture vessel; (b) a volume of growth mediumwithin said vessel sufficient to fill only a portion of the volume ofsaid vessel; (c) air within said vessel that fills a portion of saidvessel adjoining said medium; (d) a porous substrate located at theinterface between said medium and said air; and (d) cells expressingmarkers characteristic of pancreatic endoderm/endocrine precursor cellsderived from pluripotent stem cells disposed upon the surface of saidsubstrate such that said medium contacts only a portion of the surfaceof said cells.

In one embodiment, Stage 6 cells cultured according to embodiments ofthe invention are utilized and differentiated into Stage 7 cells, whilein other embodiments Stage 6 cells cultured according to other protocolsmay be utilized.

In another embodiment, the methods of the invention result in theformation of Stage 7 cells that are single-hormone positive. Thus, inone embodiment, the methods of the invention result in Stage 7 cells,which co-express NKX6.1, insulin, PDX1, and MAFA. In another embodiment,the methods of the invention result in Stage 7 cells, which co-expressNKX6.1, PDX1, insulin and MAFA. In still another embodiment, apopulation of cells in which each of the cells of at least about 10%,alternatively at least about 20%, alternatively at least about 30%,alternatively at least about 40%, alternatively at least about 50%,alternatively at least about 60%, alternatively at least about 70%,alternatively at least about 80%, or alternatively at least about 90% ofthe cell population express insulin, PDX1. NKX6.1, and MAFA result.

In certain and preferred embodiments of the invention, the methodemploys BLAR a custom medium (Table I) at Stages 4 through 7 or lateStage 4 through 7, or Stages 5, 6 and 7. The medium may preferably beexchanged every day or alternatively every other day. In anotherembodiment, the invention relates to a method of forming Stage 7 cellsco-expressing NKX6.1, PDX1, MAFA and insulin comprising culturing Stage4, preferably late Stage 4 cells to Stage 7 cells at the air-liquidinterface. In yet another embodiment, the invention relates to a methodof forming single hormone insulin positive cells expressing NKX6.1,PDX1, and MAFA Stage 7 cells by culturing Stage 4, preferably late Stage4 cells, to Stage 7 cells at the air-liquid interface.

Stage 6 and 7 cells generated according to the methods described hereinare also well-suited for use in screening compounds for their effect onthe secretion of pancreatic hormones and endocrine markers. Inparticular, Stage 4 through Stage 7 cells cultured at ALI can be testedin different culture formats from 384 to 6-well formats. Such formatsallow for evaluation of a variety of small molecules or biologics atvarious doses and time intervals on subsequent expression of pancreaticendoderm, pancreatic endocrine precursor, pancreatic endocrine, andpancreatic beta cell markers. Such an evaluation may be accomplished bymeasuring gene expression by PCR, protein expression by FACS or immunestaining, or by ELISA for secretion of factors by cells affected byaddition of small molecules/biologics.

Cells Obtainable by the Methods of the Invention.

The invention provides a cell, or population of cells obtainable by amethod of the invention. The invention also provides a cell, or cellpopulation, preferably expressing markers characteristics of pancreaticendocrine cells of a mature phenotype. The invention also provides aninsulin positive cell or population of insulin positive cells,preferably expressing markers characteristic of pancreatic endocrinecells of a mature phenotype, characterized by NKX6.1 expression(preferably greater than about 30%), PDX1 expression (preferably greaterthan about 30%), and MAFA expression (preferably greater than about10%).

Methods for Treatment.

The invention provides methods of treatment and in particular fortreating patients suffering from, or at risk of developing, diabetes.The invention also provides a population of cells obtainable or obtainedby a method of the invention for use in a method of treatment. Inparticular the invention provides a cell or population of cellsobtainable or obtained by a method of the invention for use in a methodof treating a person suffering from, or at risk, of developing diabetes.The diabetes may be Type 1 or Type 2.

In one embodiment, the method of treatment comprises implanting cellsobtained or obtainable by a method of the invention into a patient. Inone embodiment, the method of treatment comprises differentiatingpluripotent cells in vitro into Stage 1, Stage 2, Stage 3, Stage 4,Stage 5, Stage 6 or Stage 7 cells, for example as described herein, andimplanting the differentiated cells into a patient. In anotherembodiment, the method further comprises the step of culturingpluripotent stem cells, for example as described herein, prior to thestep of differentiating the pluripotent stem cells. In a still furtherembodiment, the method further comprises the step of differentiating thecells in vivo after the step of implantation. In one embodiment, thepatient being treated by any of the methods is a mammal and preferablyis a human.

In one embodiment, the cells may be implanted as dispersed cells orformed into clusters that may be infused into the vascular system, forexample, the hepatic portal vein. Alternatively, the cells may beprovided in a biocompatible, degradable, polymeric support, porous,non-degradable devices, or encapsulated to protect from the immunesystem of the host. In one embodiment, the method of treatment furthercomprises incorporating cells into a three-dimensional support prior toimplantation. The cells can be maintained in vitro on this support priorto implantation into the patient. Alternatively, the support containingthe cells can be directly implanted in the patient without additional invitro culturing. The support can optionally be incorporated with atleast one pharmaceutical agent that facilitates the survival andfunction of the transplanted cells. Cells may be implanted into anappropriate site in a recipient including, for example, the liver,natural pancreas, renal subscapular space, omentum, peritoneum,subserosal space, intestine, stomach, or a subcutaneous pocket.

To enhance further differentiation, survival or activity of theimplanted cell in vivo, additional factors, such as growth factors,antioxidants, or anti-inflammatory agents may be administered before,simultaneously with, or after administration of the cells. These factorscan be secreted by endogenous cells and exposed to the administeredcells in situ. Implanted cells can be induced to differentiate by anycombination of endogenous and exogenously administered growth factorsknown in the art.

The amount of cells used in implantation depends on a number of factorsincluding the condition of the implantation subject and response to theimplanted therapy and can be determined by one skilled in the art.

The invention will be further clarified by a consideration of thefollowing, non-limiting examples.

EXAMPLES Example 1 Pancreatic Endoderm Cells Cultured at the Air-LiquidInterface—Progressive Increase in Expression of MAFA from Stage 5 toStage 7

This example demonstrates the kinetics of MAFA expression in pancreaticendoderm cells cultured at an air-liquid interface (“ALI”) during Stages5 through 7 and treated from Stage 5 to Stage 7 with T3 and ALK5inhibitor II. Cells of the human embryonic stem cell line H1 (WA01cells, WiCell Research Institute, Madison, Wis.) at passage 42 wereseeded as single cells at 1×10⁵ cells/cm² on MATRIGEL™ at a 1:30dilution (Corning Incorporated, Corning N.Y., Catalog No. 354230) coateddishes in a media of Dulbecco's Modified Eagle's Medium; Nutrientmixture F-12 (“DMEM-F12”) (Life Technologies Corporation, Carlsbad,Calif., Catalog No. 11330-032), GlutaMAX™ (Life Technologies, CatalogNo. 35050-079) in a 1:100 dilution (“1× concentration”), 0.25 mMascorbic acid (Sigma Aldrich Co. LLC, St. Louis Mo., Catalog No. A4544),100 ng/ml of fibroblast growth factor 2 (“FGF2”) (R & D Systems,Minneapolis, Minn., Catalog No. 233-FB-025), 1 ng/ml of transforminggrowth factor beta (“TGF-β”) (R & D Systems Inc., Catalog No.240-B-002), insulin-transferrin-selenium-ethanolamine (“ITS-X”) (LifeTechnologies, Catalog No. 51500056) at a 1:100 dilution, 2% fatty-acidfree bovine serum albumin (“FAF-BSA”) (Proliant, Inc., Boone, Idaho,Catalog No. 68700), and 20 ng/ml of insulin-like growth factor-1(“IGF-1”) (R & D Systems, Catalog No. 291-G1-200), supplemented with 10μM of Rock inhibitor Y-27632 (Catalog No. Y0503, Sigma-Aldrich).Forty-eight hours post-seeding, the cultures were washed in incompletePBS (phosphate buffered saline without magnesium or calcium) followed byincubation with 1× TrypLE™ Express Enzyme (Life Science; Catalog No.14190) for 3 to 5 minutes at 37° C. The released cells were rinsed withDMEM-F12 and spun at 1000 rpm for 5 minutes. The resulting cell pelletwas resuspended in DMEM-F12 supplemented with 10 μM Y-27632, GlutaMAX™in a 1:100 dilution (“1× concentration”), 0.25 mM ascorbic acid, 100ng/ml FGF2, 1 ng/ml TGF-β, ITS-X at a 1:100 dilution, 2% FAF-BSA and 20ng/ml IGF-1 and the single cell suspension was seeded at approximately1.3 to 1.5×10⁵ cells/cm². The cultures were fed every day with mediumand differentiation, according to the following protocol, was initiated48 hrs. following seeding resulting in an about 90% starting confluency.During Stages 1 through 4 of the differentiation protocol used, cultureswere maintained on planar adherent cultures and at the air-liquidinterface for Stages 5 through 7.

Stage 1 (3 Days):

Cells were plated on MATRIGEL™ (1:30 dilution)-coated dishes were firstrinsed with 1× incomplete DPBS and then were cultured for one day in thefollowing Stage 1 media: MCDB-131 medium (Life Technologies, Catalog No.10372-019) supplemented with 0.5% FAF-BSA, 1.2 g/1000 ml sodiumbicarbonate (Sigma-Aldrich Catalog No. S3187); GlutaMAX™ at aconcentration of 1×; 4.5 mM D-glucose (Sigma-Aldrich, Catalog No. G8769)in this stage and the following stages where used, to obtain aconcentration of 10 mM of glucose (Sigma-Aldrich, Catalog No. G8769);100 ng/ml growth/differentiation factor 8 (“GDF8”) (Peprotech, RockyHill, N.J. Catalog No. 120-00); and 1 μM of a14-Prop-2-en-1-yl-3,5,7,14,17,23,27-heptaazatetracyclo[19.3.1.1˜2,6˜.1˜8,12˜]heptacosa-1(25),2(27),3,5,8(26),9,11,21,23-nonaen-16-one(“MCX compound”). Cells were then cultured for an additional day inMCDB-131 medium supplemented with 0.5% FAF-BSA, 0.0012 g/ml sodiumbicarbonate, 1× concentration of GlutaMAX™, 4.5 mM D-glucose, 100 ng/mlGDF8, and 0.1 μM MCX compound. Cells were then cultured for anadditional day in MCDB-131 medium supplemented with 0.5% fatty acid-freeBSA, 1.2 g/1000 ml sodium bicarbonate, 1× GlutaMAX™, 4.5 mM D-glucose,and 100 ng/ml GDF8.

Stage 2 (2 Days):

Cells were first rinsed with 1× incomplete DPBS and then were treatedfor two days with MCDB-131 medium supplemented with 0.5% FAF-BSA; 1.2g/1000 ml sodium bicarbonate; 1× GlutaMAX™; 4.5 mM D-glucose; 0.25 mMascorbic acid and 25 ng/ml FGF7 (R & D Systems, Inc., Catalog No.251-KG.).

Stage 3 (2 Days):

Cells were treated with BLAR custom medium (manufactured by LifeTechnologies, components listed on Table I) supplemented with a 1:200dilution of ITS-X; 4.5 mM glucose; 1× GlutaMAX™; 2.5 g/1000 ml sodiumbicarbonate; 2% FAF-BSA; 0.25 μM SANT-1(N-[(3,5-dimethyl-1-phenyl-1H-pyrazol-4-yl)methylene]-4-(phenylmethyl)-1-piperazinamine)(Sigma Aldrich, Catalog No. S4572); 1 μM retinoic acid (“RA”) (SigmaAldrich, Catalog No. R2625); 25 ng/ml FGF7; 0.25 mM ascorbic acid; 200nM the PKCactivator ((2S,5S-(E,E)-8-(5-(4-trifluoromethyl)phenyl-2,4,-pentadienoylamino)benzolactam(“TPB”) (EMD Millipore Corporation, Gibbstown, N.J.; Catalog No.565740); and 100 nM of the bone morphogenic protein (“BMP”) receptorinhibitor LDN-193189 (Shanghai ChemPartners Co Ltd., Shanghai, China)for two days.

Stage 4 (3 Days):

Cells were treated with BLAR medium supplemented with a 1:200 dilutionof ITS-X; 4.5 mM glucose; 1× concentration of GlutaMAX™; 2.5 g/1000 mlsodium bicarbonate; 2% FAF-BSA; 0.25 μM SANT-1; 100 nM RA; 2 ng/ml FGF7;100 nM LDN-193189; 0.25 mM ascorbic acid; and 200 nM TPB for three days.At end of Stage 4 (3 days), cells cultured on planar dishes were treatedfor 4 hours with 10 μM of Y27632, rinsed with PBS and treated for 5minutes at room temperature with the enzyme TrypLE™ Express Enzyme(LifeTechnologies Corporation, Catalog No. 12604-013) at a concentrationof 1× followed by removal of the enzyme, rinsing with BLAR media andscraping of cells by a cell scraper. The resulting suspension of cellswere seeded at a density of 0.5-0.75×10⁶ cells (in 5-10 μl aliquots) on0.4 micron porous cell culture filter inserts (Corning, Catalog No.353493) in 6-well plates. 1.5 ml of media was added to the bottom ofeach insert and no further media was added to the apical, or top, sideof the filter. The media was replaced daily for the duration of Stages5, 6 and 7.

Stage 5 (3 Days):

Cells cultured at the air-liquid interface were treated with BLAR mediumsupplemented with a 1:200 dilution of ITS-X; glucose to achieve a finalconcentration of 20 mM glucose; 1× GlutaMAX™; 1.5 g/1000 ml sodiumbicarbonate; 2% FAF-BSA; 0.25 mM ascorbic acid; 10 μg/ml of heparin(Sigma Aldrich, Catalog No. H3149), 10 μM ZnSO₄ (Sigma Aldrich, CatalogNo. Z0251), 0.25 μM SANT-1; 50 nM RA; 100 nM LDN-193189; 1 μM of T3 inthe form of 3,3′,5-triiodo-L-thyronine sodium salt (Sigma Aldrich,Catalog No. T6397), 10000 nM of2-(3-(6-Methylpyridin-2-yl)-1H-pyrazol-4-yl)-1,5-naphthyridine (“ALK5inhibitor II”) (Enzo Life Sciences, Inc., Farmingdale, N.Y., Catalog No.ALX-270-445) for three days.

Stage 6 (7 Days):

Stage 5 cells were treated with BLAR medium supplemented with a 1:200dilution of ITS-X; glucose to achieve a final concentration 20 mMGlucose; 1× concentration of GlutaMAX™; 1.5 g/ml sodium bicarbonate; 2%FAF-BSA; 0.25 mM ascorbic acid; 10 μg/ml of heparin, 10 μM ZnSO₄, 100 nMLDN-193189, 1 μM T3, 100 nM(S,S)-2-[2-(3,5-Difluorophenyl)acetylamino]-N-(5-methyl-6-oxo-6,7-dihydro-5H-dibenzo[b,d]azepin-7-yl)propionamide(“gamma secretase inhibitor XX”) (EMD Millipore, Catalog No. #565789),and 10000 nM ALK5 inhibitor II for 7 days.

Stage 7 (7 Days):

Stage 6 cells were treated with BLAR medium supplemented with a 1:200dilution of ITS-X; glucose to achieve a final concentration 20 mMGlucose, 1× GlutaMAX™; 1.5 g/1000 ml sodium bicarbonate; 2% FAF-BSA; 10μg/ml of heparin, 10 μM ZnSO₄, 1 μM T3, 10000 nM ALK5 inhibitor II, 10μM the vitamin E analogue Trolox (EMD Millipore Catalog No. 648471) for7-15 days.

FIGS. 1A, 1B, 1C, 1D, 1E, 1F, 1G, 1H, 1I, 1J, 1K, 1L, and 1M depict datafrom real-time PCR analyses of the following genes in cells of the humanembryonic stem cell line H1 differentiated as outlined in Example 1:PDX1 (FIG. 1A); NKX6.1 (FIG. 1B); PAX4 (FIG. 1C); PAX6 (FIG. 1D); NGN3(FIG. 1E); MAFA (FIG. 1F); ABCC8 (FIG. 1G); chromogranin-A (FIG. 1H);G6PC2 (FIG. 1I); IAPP (FIG. 1J); insulin (FIG. 1K); glucagon (FIG. 1L);and PTF1a (FIG. 1M). As shown in FIG. 1F, there was a clear increase, orupregulation, in MAFA comparing Stages 4 and 5 to Stages 6 and 7demonstrating an increased maturation of cells towards a beta celllineage. However, at Stages 6 and 7, the mRNA expression for MAFA waslower than adult human islets.

TABLE I List of components of BLAR media Component Concentration (mM)Amino Acids Glycine 3.0E−02 Alanine 3.0E−02 Arginine 3.0E−01 Aspargine1.0E−01 Aspartic Acid 1.0E−01 Cysteine 2.0E−01 Glutamic acid 3.0E−02Histidine 1.1E−01 Isoleucine 1.0E−02 Leucine 9.0E−02 Lysinehydrochloride 1.5E−01 Methiane 3.0E−02 Phenylalanine 3.0E−02 Proline1.0E−01 Serine 1.0E−01 Theronine 3.0E−02 Tryptophan 2.0E−03Tyrosinedisodium 1.0E−02 Valine 3.0E−02 Vitamins Biotin 3.0E−05 Cholinechloride 5.0E−03 D-Calcium pantothenate 1.5E−03 Folinic Acid Calciumsalt 2.3E−03 Niacinamide 4.9E−03 Pyridoxine hydrochloride 9.7E−04Riboflavin 1.0E−05 Thiamine hydrochloride 3.0E−03 Vitamin B12 3.7E−06i-Inositol 2.8E−03 Minerals/other Calcium Chloride (CaCl₂—2H₂O) 3.0E−01Cupric sulfate (CuSO₄—5H2O) 4.8E−06 Ferric sulfate (FeSO₄—7H₂O) 1.0E−03Magnesium Sulfate (MgSO4—7H₂O) 4.1E−01 Potassium Chloride (KCl) 3.8E+00Sodium Bicarbonate (NaHCO₃) 1.4E+01 Sodium Chloride (NaCl) 1.1E+02Sodium Phosphate dibasic (Na2HPO4—7H₂O) 5.0E−01 Zinc Sulfate (ZnSO₄—H₂O)1.0E−04 Adenine 1.0E−03 D-Glucose (Dextrose) 5.0E+00 Lipoic Acid 1.2E+05Phenol Red 1.0E−02 Sodium Pyruvate 1.0E+00 Thymidine 9.8E−05

For additional characterization of various stages, cells were harvestedat Stages 3, 4, 5, 6, and 7 and analyzed by fluorescence-activated flowcytometry (“FACS”). FACS staining was conducted as described inDiabetes, 61, 2016, 2012 and using the antibodies listed in Table III.In brief, cells were incubated in TrypLE™ Express (Life Technologies,Catalog No. 12604) for 3-5 minutes at 37° C. and released into singlecell suspensions after which they were washed twice with a stainingbuffer of PBS containing 0.2% BSA (BD Sciences, Catalog No. 554657).Cells (1×10⁵ to 1×10⁶) were re-suspended in 100 μl blocking buffer of0.5% human gamma globulin diluted 1:4 in staining buffer for surfacemarking. Added to the cells at a final dilution of 1:20 were directlyconjugated primary antibodies followed by incubation at 4° C. for 30minutes. The stained cells were twice washed in the staining buffer,followed by re-suspension in 200 μl staining buffer and then incubatedin 15 μl of 7AAD for live/dead discrimination before FACS analysis onthe BD Canto II. Intracellular antibody staining was accomplished byfirst incubating with Green Fluorescent LIVE/DEAD cell dye (LifeTechnologies, Catalog No. L23101) at 4° C. for 20 minutes followed by asingle wash in cold PBS. Fixing of cells was in 250 μl ofCytofix/Cytoperm Buffer (BD Catalog No. 554723) followed byre-suspension of the cells in 100 μl of Perm wash bufferstaining/blocking solution with 2% normal goat serum. Cells wereincubated at 4° C. for 30 minutes with primary antibodies at empiricallypre-determined dilutions followed by two washes in Perm/Wash buffer.Cells were then incubated with the appropriate antibodies at 4° C. for30 minutes and then washed twice prior to analysis on a BD FACS CantoII. The concentrations of antibodies used are shown on Table III. Theantibodies for pancreas markers were tested for specificity using humanislets or undifferentiated H1 cells as a positive control. For secondaryantibodies, the following were added and incubated at 4° C. for 30minutes: anti-mouse Alexa Fluor® 647 at 1:500 (Life Technologies), goatanti-rabbit PE at 1:200 (v) or donkey anti-goat Alexa 647 at 1:800 (LifeTechnologies) followed by a final wash in Perm/Wash buffer and analysison a BD FACS Canto II using BD FACS Diva Software with at least 30,000events being acquired.

FIGS. 2A, 2B, 2C, 3A, 3B, 3C, 3D, 4A, 4B, 4C, 4D, 4E, 5A, 5B, 5C, 5D,5E, 5F, 6A, 6B, 6C, 6D, 6E and 6F depict FACS profiles of cellscollected at Stages 3, 4, 5, 6, and 7, respectively. As shown in FIGS.4A, 4B, 4C, 4D and 4E, at Stage 5, approximately 15% of cells wereco-expressing insulin and NKX6.1 and approximately 21% of PDX1 positivecells were in active cell cycle as measured by co-expression of PDX1 andKI-67 (˜23%; KI-67 is indicative of cells that are in active cellcycle). However, by Stage 6 and Stage 7 (FIGS. 5A, 5B, 5C, 5D, 5E, 5F,6A, 6B, 6C, 6D, 6E and 6F), there was a significant drop inproliferating PDX1+ cells (1-2%) while there was a significant increasein the number of NKX6.1+ cells co-expressing insulin (˜39% at Stage 6and ˜50% at Stage 7). Moreover, there was a significant rise in cellsexpressing endocrine precursor markers ISL-1, NeuroD1, and NKX2.2. Theseresults indicate that the cultures of Stages 6 and 7 allowed for rapidmaturation of cells away from a proliferating progenitor fate to earlymaturing endocrine cells. In addition, an increase in the percentage ofcells co-expressing insulin and NXK6.1 (33%) was observed by prolonginggoing from Stage 6 to Stage 7 (FIGS. 5A, 5B, 5C, 5D, 5E, 5F as comparedto FIGS. 6A, 6B, 6C, 6D, 6E and 6F). FIG. 7 summarizes the percentexpression of multiple pancreatic endoderm (FOXA2, PDX-1, NKX6.1),undifferentiated ES cells (Oct3/4), endocrine (Pax6, IS1-1, NKX2.2,chromogranin), and hormone (insulin, glucagon) from Stage 3 throughStage 7.

TABLE II List of Antibodies used for FACS analysis Dilu- Antigen SpeciesSource/Catalogue Number tion Glucagon Mouse Sigma-Aldrich Co. LLC/G2654 1:250 Insulin Rabbit Cell Signaling Technology. Inc., 1:10 Danvers.MA/3014B NKX6.1 Mouse Developmental Studies Hybridoma 1:50 Bank. IowaCity, Iowa/F55A12 NKX2.2 Mouse Developmental Studies Hybridoma  1:100Bank/74.5A5 PDX1 Mouse BD BioSciences, San Jose, CA/ 1:50 562161 Ki67Mouse BD Biosciences/558595 1:20 Pax6 Mouse BD Biosciences, 561552 1:20Chromogranin A Rabbit Dako, Carpinteria, CA/A0430 1:40 ISL-1 Mouse BDBiosciences/562547 1:20 NeuroD1 Mouse BD Bioscience/563001 1:40 FOXA2Mouse BD Bioscience/561589 1:80 OCT3/4 Mouse BD Biosciences/560329 1:20

Example 2 Screening to Identify Small Molecules that can SignificantlyUpregulate One or Both of MAFA and Insulin Expression

This example is directed to identify small molecules that cansignificantly enhance maturation of cells towards a pancreatic betacell. Cells of the human embryonic stem cell line H1 (WA01) at passage42 were seeded as single cells at 1×10⁵ cells/cm² on MATRIGEL™ (1:30dilution)-coated dishes in a media comprising of DMEM-F12, GlutaMAX™(1:100 dilution), 0.25 mM ascorbic acid, 100 ng/ml of FGF2 (R & Dsystems, MN), 1 ng/ml of TGF-β, ITS-X (1:100 dilution), 2% FAF-BSA, and20 ng/ml of IGF-1, supplemented with 10 μM of Y-27632. Forty-eight hourspost-seeding, the cultures were washed in incomplete PBS (phosphatebuffered saline without Mg or Ca) followed by incubation with 1× TrypLE™Express Enzyme (Life Science; Catalog No. 14190) for 3 to 5 minutes at37° C. The released cells were rinsed with DMEM-F12 and spun at 1000 rpmfor 5 minutes. The resulting cell pellet was resuspended in DMEM-F12supplemented with 10 μM Y-27632, 1× GlutaMAX™, 0.25 mM ascorbic acid,100 ng/ml FGF2, 1 ng/ml TGF-β, ITS-X at a 1:100 dilution, 2% FAF-BSA and20 ng/ml of IGF-1 and the single cell suspension was seeded atapproximately 1.3 to 1.5×10⁵ cells/cm². The cultures were fed every daywith medium and differentiation, according to the following protocol,was initiated 48 hrs. following seeding resulting in an about 90%starting confluency. Unless otherwise stated, the sources for the media,reagents, molecules and the like used in the examples are as stated inExample 1.

Stage 1 (3 Days):

Cells were plated on MATRIGEL™ (1:30 dilution)-coated dishes were firstrinsed with 1× incomplete DPBS and then were cultured for one day inStage 1 media: MCDB-131 medium supplemented with 0.5% FAF-BSA, 0.0012g/ml sodium bicarbonate; 1× concentration of GlutaMAX™; 4.5 mMD-glucose; 100 ng/ml GDF8; and 1 μM MCX compound. Cells were thencultured for an additional day in MCDB-131 medium supplemented with 0.5%FAF-BSA, 0.0012 g/ml sodium bicarbonate, 1× concentration of GlutaMAX™,4.5 mM D-glucose, 100 ng/ml GDF8, and 0.1 μM MCX compound. Cells werethen cultured for an additional day in MCDB-131 medium supplemented with0.5% FAF-BSA, 0.0012 g/ml sodium bicarbonate, 1× concentration ofGlutaMAX™, 4.5 mM D-glucose, and 100 ng/ml GDF8.

Stage 2 (2 Days):

Cells were first rinsed with 1× incomplete DPBS and then were treatedfor two days with MCDB-131 medium supplemented with 0.5% FAF-BSA; 0.0012g/ml sodium bicarbonate; 1× concentration of GlutaMAX™; 4.5 mMD-glucose; 0.25 mM ascorbic acid and 25 ng/ml FGF7.

Stage 3 (2 Days):

Cells were treated with BLAR custom medium supplemented with a 1:200dilution of ITS-X; 4.5 mM glucose; 1× concentration of GlutaMAX™; 0.0025g/ml sodium bicarbonate; 2% FAF-BSA; 0.25 μM SANT-1; 1 μM RA; 25 ng/mlFGF7; 0.25 mM ascorbic acid; 200 nM TPB; and 100 nM LDN-193189 for twodays.

Stage 4 (3 Days):

Cells were treated with BLAR medium supplemented with a 1:200 dilutionof ITS-X; 4.5 mM glucose; 1× concentration of GlutaMAX™; 0.0025 g/mlsodium bicarbonate; 2% FAF-BSA; 0.25 μM SANT-1; 100 nM RA; 2 ng/ml FGF7;100 nM LDN-193189; 0.25 mM ascorbic acid; and 200 nM TPB for three days,then at end of Stage 4 cells cultured on planar dishes were treated for4 hours with 10 μM of Y-27632, rinsed with PBS and treated for 5 minutesat room temperature with 1× concentration of TrypLE™ followed by removalof the enzyme, rinsing with BLAR basal media and scraping of cells by acell scraper. The resulting suspension of cells were seeded at a densityof 0.5-0.75×10⁶ cells (in 5-10 μl aliquots) on 0.4 micron porous cellculture filter inserts in 6-well plates. 1.5 ml of media was added tothe bottom of each insert and no further media was added to the apicalside of the filter. The media was replaced daily for the duration ofStages 5, 6 and 7.

Stage 5 (3 Days):

Cells cultured at the air-liquid interface were treated with BLAR mediumsupplemented with a 1:200 dilution of ITS-X; 20 mM glucose; 1×GlutaMAX™; 0.0015 g/ml sodium bicarbonate; 2% FAF-BSA; 0.25 mM ascorbicacid; 10 μg/ml of heparin, 10 μM ZnSO₄, 0.25 μM SANT-1; 50 nM RA; 100 nMLDN-193189; 1 μM of T3 as 3,3′,5-triiodo-L-thyronine sodium salt, and10000 nM of ALK5 inhibitor II for three days.

Stage 6 (7 Days):

Stage 5 cells were cultured at the air-liquid interface and treated withBLAR medium supplemented with a 1:200 dilution of ITS-X; 20 mM Glucose;1× concentration of GlutaMAX™; 0.0015 g/ml sodium bicarbonate; 2%FAF-BSA; 0.25 mM ascorbic acid; 10 μg/ml of heparin, 10 μM ZnSO₄, 100 nMLDN-193189, 1 μM T3 as 3,3′,5-triiodo-L-thyronine sodium salt, 100 nMgamma secretase inhibitor XX, and 10000 nM ALK5 inhibitor II for 7 days.

Stage 7 (7 Days):

Stage 6 cells were cultured at the air-liquid interface and treated withBLAR medium supplemented with a 1:200 dilution of ITS-X; 20 mM Glucose;1× concentration of GlutaMAX™; 0.0015 g/ml sodium bicarbonate; 2%FAF-BSA; 10 μg/ml of heparin, 10 μM ZnSO₄, 1000 nM T3 as3,3′,5-triiodo-L-thyronine sodium salt, 10000 nM ALK5 inhibitor II, 10μM Trolox for 7 days

At Stages 6 or 7, various small molecules were added and their effectwas evaluated by real-time PCR. Table III lists the small moleculesevaluated and when they were added.

TABLE III Small molecules evaluated Small Molecule Chemical name/CAS#/Molecular formula Target Name/Vendor/Catalog No. Concentration4-(Acetylamino)-N-(2- histone deacetylase (HDAC) CI-994/Sigma Aldrich Co1 μM at S6 aminophenyl)benzamide/112522-64- inhibitor LLC./EPI109A2/C₁₅H₁₅N₃O₂ (E)-N-hydroxy-3-[4-[[2-(2-methyl-1H-indol-3- inhibitor ofboth histone Panobinostat (LBH-589)/Sigma 1 μM at S6yl)ethylamino]methyl]phenyl]prop-2- deacetylase 1 (HDAC1) Aldrich CoLLC./EPI009B enamide/404950-80-7/C₂₁H₂₃N₃O₂ activityN-hydroxy-N′-phenyl-octanediamide/149647- inhibitor of histoneSAHA/Sigma Aldrich Co 1 μM at S6 78-9/C₁₄H₂₀N₂O₃ deacetylase 1 (HDAC1)LLC./EPI009C and 3 (HDAC3) N,N′-Dihydroxyoctanediamide/38937-66- Histonedeacetylase (HDAC) SBHA/Sigma Aldrich Co 1 μM at S6 5/C₈H₁₆N₂O₄inhibitor that has been LLC./EPI009D shown to inhibit HDAC16-(1,3-Dioxo-1H,3H-benzo[de]isoquinolin-2- Inhibitor of histoneScriptaid/Sigma Aldrich Co 1 μM at S6 yl)-hexanoic acid hydroxyamide/deacetylase (HDAC) LLC./EPI009E 287383-59-9/C₁₈H₁₈N₂O₄[R-(E,E)]-7-[4-(Dimethylamino)phenyl]-N- Inhibitor of histoneTrichostatin A/Sigma Aldrich Co 1 μM at S6hydroxy-4,6-dimethyl-7-oxo-2,4- deacetylase LLC./EPI009Fheptadienamide/58880-19-6/C₁₇H₂₂N₂O₃N1-[4-[(2R,4R,6S)-4-[[(4,5-diphenyl-2- Inhibitor of histoneTubacin/Sigma Aldrich Co 1 μM at S6 oxazolyl)thio]methyl]-6-[4-deacetylase 6 LLC./EPI009G (hydroxymethyl)phenyl]-1,3-dioxan-2-yl]phenyl]-N8-hydroxy-octanediamide/ 537049-40-4/C₄₁H₄₃N₃O₇S1,4-Dimethoxy-9(10H)- Cyclin-dependent kinase NSC625987/Tocris 1 μM atS6 acridinethione/141992-47-4/C₁₅H₁₃NO₂S (cdk) 4 inhibitorBioscience/2152 4-[4,5-Dihydro-5-(4-methoxyphenyl)-3- Inhibitor of Cdc42GTPase ML141/Tocris Bioscience/4266 1 μM at S6 phenyl-1H-pyrazol-1-yl]benzenesulfonamide/71203-35- 5/C₂₂H₂₁N₃O₃SN-(2-chlorophenyl)-6-(piperidin-4- Inhibitor of EphB3 receptor LDN211904(EphB3 1 μM at S6 yl)imidazo[1,2-a]pyridine-3- tyrosine kinaseinhibitor)/EMD Millipore carboxamide oxalate/1198408-39- Corporation,Billerica, 7/C₂₁H₂₁ClN₄O₅ MA/428201-5MG(S)-2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H- CPIe inhibitor of theCPI203/Xcess Biosciences, Inc., 1 μM at S6thieno[3,2-f][1,2,4]triazolo[4,3- bromodomain and extra San Diego,CA/M60124-2 a][1,4]diazepin-6-yl)acetamide/1446144- terminal (BET)family 04-2/C₁₉H₁₈ClN₅OS protein BRD41-(3,6-dibromo-9H-carbazol-9-yl)-3- pro-neurogenic, neuro- P7C3/XcessBiosciences, 1 μM at S6 (phenylamino)propan-2-ol/301353-96- protectivesmall Inc./M60017-2 8/C₂₁H₁₈Br₂N₂O molecule2-(4-benzoylphenoxy)-N-(1-benzylpiperidin- agonist of adiponectinAdipoRon/Xcess Biosciences, 1 μM at S64-yl)acetamide/924416-43-3/C₂₇H₂₈N₂O₃ receptor (AdipoR) Inc./M60152-2s(S)-2-((S)-2-(3,5-difluorophenyl)-2- inhibitor of gamma secretaseLY411575/Xcess Biosciences, 1 μM at S6hydroxyacetamido)-N-((S)-5-methyl-6- Inc./M60078-5soxo-6,7-dihydro-5H-dibenzo[b,d]azepin- 7-yl)propanamide/209984-57-6/C₂₆H₂₃F₂N₃O₄ 2-(4-(tert-butyl)phenyl)1H- transcriptional activator ofZLN005/Xcess Biosciences, 1 μM at S6benzo[d]imidazole/49671-76-3/C₁₇H₁₈N₂ PGC-1α Inc./M60142-5s2-chloro-4-fluoro-3-methyl-N-(2-(4- antagonist of WDR5-MLLWDR5-C47/Xcess Biosciences, 1 μM at S6 methylpiperazin-1-yl)-5-interaction Inc./M60118-2 nitrophenyl)benzamide/1422389-91-0/C₁₉H₂₀ClFN₄O₃ 3-pyridinylmethyl [[4-[[(2-aminophenyl) HDAC inhibitor;MS-275/Sigma Aldrich Co 1 μM at S6 amino]carbonyl]phenyl]methyl]antiproliferative; LLC./EPS002 carbamate/209783-80-2/C₂₁H₂₀N₄O₃Preferentially inhibits HDAC1 over HDAC34-(Dimethylamino)-N-[7-(hydroxyamino)-7- HDAC inhibitor; subtypeM344/Sigma Aldrich Co 1 μM at S6 oxoheptyl]-benzamide/251456-60-selective for HDAC6 LLC./M5820 7/C₁₆H₂₅N₃O₃ over HDAC11-[[4-Ethyl-5-[5-(4-phenoxyphenyl)-1,2,4- Sphingosine-1-phosphateCS2100/Tocris Bioscience, 1 μM at S6oxadiazol-3-yl]-2-thienyl]methyl]-3- receptor 1 (S1P1) Bristol, BS110QL, UK/ azetidinecarboxylic acid/913827-99- agonist; Exhibits 5000-4543 3/C₂₅H₂₃N₃O₄S fold selectivity for human S1P1 over S1P32-(4-Bromo-2-chlorophenoxy)-N-[[[4-[[(4,6- Selective inhibitor of Cdc42ZCL278/Tocris Bioscience/4794 1 μM at S6 dimethyl-2-pyrimidinyl)amino]sulfonyl]phenyl]ami-no]thioxomethyl]acetamide/587841-73- 4/C₂₁H₁₉BrClN₅O₄S₂N-(4-(2-amino-3-chloropyridin-4-yloxy)-3- Met-related inhibitor for c-BMS-777607/Selleck Chemicals, 1 μM at S6 fluorophenyl)-4-ethoxy-1-(4-Met, Axl, Ron and Houston, TX/S1561 fluorophenyl)-2-oxo-1,2- Tyro3dihydropyridine-3- carboxamide/1025720-94- 8/C₂₅H₁₉ClF₂N₄O₄N-(2,6-difluorophenyl)-5-(3-(2-(5-ethyl-2- Inhibitor of IGF-1R and IRGSK1904529A/Selleck 1 μM at S6 methoxy-4-(4-(4- Chemicals/S1093(methylsulfonyl)piperazin-1- yl)piperidin-1-yl)phenylamino)pyrimidin-4-yl)H- imidazo[1,2-a]pyridin-2-yl)-2-methoxybenzamide/1089283-49- 7/C₄₄H₄₇F₂N₉O₅SN-(4-(3-(2-aminopyrimidin-4-yl)pyridin-2- pan-Aurora kinases inhibitorAMG-900/Selleck 1 μM at S6 yloxy)phenyl)-4-(4-methylthiophen-2- forAurora A/B/C Chemicals/S2719 yl)phthalazin-1-amine/945595-80-2/C₂₈H₂₁N₇OS 5-((1-(3-isopropyl-1,2,4-oxadiazol-5- GPR119 agonistGSK1292263/Selleck 1 μM at S6 yl)piperidin-4-yl)methoxy)-2-(4-Chemicals/S2149 (methylsulfonyl)phenyl)pyridine/1032823-75-8/C₂₃H₂₈N₄O₄S 4-(5-Amino-1-(2,6-difluorobenzoyl)-1H- pan-CDKinhibitor with the JNJ-7706621/Selleck 1 μM at S6[1,2,4]triazol-3-ylamino)- highest potency on Chemicals/S1249benzenesulfonamide/443797-96- CDK1/2 4/C₁₅H₁₂F₂N₆O₃S6-(difluoro(6-(1-methyl-1H-pyrazol-4-yl)- inhibitor of c-MetJNJ-38877605/Selleck 1 μM at S6 [1,2,4]triazolo[4,3-b]pyridazin-3-Chemicals/S1114 yl)methyl)quinoline/943540-75- 8/C₁₉H₁₃F₂N₇(R)-4-(8-cyclopentyl-7-ethyl-5-methyl-6-oxo- Plk1 inhibitor BI2536/Selleck Chemicals/S1109 1 μM at S65,6,7,8-tetrahydropteridin-2-ylamino)-3- methoxy-N-(1-methylpiperidin-4-yl)benzamide/755038-02-9/C₂₈H₃₉N₇O₃N-hydroxy-2-(4-(((1-methyl-1H-indol-3- HDAC inhibitor with highestQuisinostat (JNJ- 1 μM at S6 yl)methylamino)methyl)piperidin-1- potencyfor HDAC1 and 26481585)/Selleck yl)pyrimidine-5-carboxamide/875320-lowest potency for Chemicals/S1096 29-9/C₂₁H₂₆N₆O₂ HDACs 6 and 7; Phase2 cyclohexyl 2,7,7-trimethyl-4-(4-nitrophenyl)- inhibitor of Notchsignaling FLI-06/Selleck Chemicals/S7399 1 μM at S65-oxo-1,4,5,6,7,8-hexahydroquinoline-3-carboxylate/313967-18-9/C₂₅H₃₀N₂O₅4H-[1,2,4]Triazolo[4,3-a][1,4]benzodiazepine- inhibitor for BET proteinsI-BET-762 (GSK525762)/Selleck 1 μM at S6 4-acetamide,6-(4-chlorophenyl)-N- Chemicals/S7189 ethyl-8-methoxy-1-methyl-, (4S)-/1260907-17-2/C₂₂H₂₂ClN₅O₂ N-(6-(4-(2-((4-((4-methylpiperazin-1- smallmolecule that promotes WS6/Xcess Biosciences, 1 μM at S6 yl)methyl)-3-pancreatic β cell Inc./M60097-2s (trifluoromethyl)phenyl)amino)-2-proliferation in rodent oxoethyl)phenoxy)pyrimidin-4- and human primaryyl)cyclopropanecarboxamide/1421227- islets 53-3/C₂₉H₃₁F₃N₆O₃1-[3-[[[(2R,3S,4R,5R)-5-(4-Amino-5-bromo- DOT1L methyltransferaseSGC0946/Selleck 1 μM at S6 7H-pyrrolo[2,3-d]pyrimidin-7-yl)-3,4-inhibitor Chemicals/S7079 dihydro-xytetrahydrofuran-2-yl]methyl](isopropyl)amino]pro-pyl]-3-[4-(2,2-dimethylethyl)phenyl]urea/—/ C₂₈H₄₀BrN₇O₄ 9H-Purin-6-amine,9-[5-deoxy-5-[[cis-3-[2-[6- inhibitor of protein EPZ5676/Selleck 1 μM atS6 (1,1-dimethylethyl)-1H-benzimidazol-2- methyltransferaseChemicals/S7062 yl]ethyl]cyclobutyl](1- DOT1Lmethylethyl)amino]-β-D-ribofuranosyl]-/ 1380288-87-8/C₃₀H₄₂N₈O₃(2R)-2-(N-(2-fluoro-4-(1,2,4-oxadiazol-3- γ-secretase inhibitor of Aβ40Avagacestat (BMS- 1 μM at S6 yl)benzyl)-4-chlorophenylsulfonamido)- andAβ42 708163)/Selleck 5,5,5-trifluoropentanamide/1146699-66-Chemicals/S1262 2/C₂₀H₁₇ClF₄N₄O₄S [1,1′-Biphenyl]-3-carboxamide,N-[(1,2- EZH2 inhibitor EPZ-6438/Selleck 1 μM at S6dihydro-4,6-dimethyl-2-oxo-3- Chemicals/S7128pyridinyl)methyl]-5-[ethyl(tetrahydro-2H-pyran-4-yl)amino]-4-methyl-4′-(4- morpholinylmethyl)-/1403254-99-8/C₃₄H₄₄N₄O₄ N-(4-(6,7-dimethoxyquinolin-4- VEGFR2 inhibitorCabozantinib (XL184, BMS- 1 μM at S6 yloxy)phenyl)-N-(4- 907351)/fluorophenyl)cyclopropane-1,1- Selleck Chemicals/S1119dicarboxamide/849217-68- 1/C₂₈H₂₄FN₃O₅3-(benzo[d]thiazol-2-yl)-6-ethyl-7-hydroxy-8- Skp2 inhibitorSKP2-C25/Xcess Biosciences, 1 μM at S6(piperidin-1-ylmethyl)-4H-chromen-4- Inc./M60136-2sone/222716-34-9/C₂₄H₂₄N₂O₃S 5-Chloro-2-[(E)-2-[phenyl(pyridin-2- Jumonjihistone demethylase JIB-04/Selleck Chemicals/S7281 1 μM at S6yl)methylidene]hydrazin-1- inihibitor yl]pyridine/199596-05-9/C17H13ClN41H-1,2,4-Triazole-3,5-diamine,1-(6,7- inhibitor of Axl R428(BGB324)/Selleck 1 μM at S7 dihydro-5H-benzo[6,7]cyclohepta[1,2-Chemicals/S2841 c]pyridazin-3-yl)-N3-[(7S)-6,7,8,9-tetrahydro-7-(1-pyrrolidinyl)-5H- benzocyclohepten-2-yl]-/1037624-75-1/C₃₀H₃₄N₈ 2-Chloro-3-[2-(2,4-dichlorophenoxy)ethoxy]- Potentsphingosine-1- CYM50260/Tocris 2 μM at S6-S76-(fluoromethyl)pyridine/1355026-60- phosphate receptor 4Bioscience/4677 6/C₁₄H₁₁Cl₃FNO₂ (S1P4) agonistN,N-Dicyclohexyl-5-cyclopropyl-3- Selective sphingosine-1-CYM5541/Tocris 2 μM at S6-S7 isoxazolecarboxamide/945128-26- phosphatereceptor 3 Bioscience/4897 7/C₁₉H₂₈N₂O₂ (S1P3) allosteric5-[4-Phenyl-5-(trifluoromethyl)thiophen-2-yl]- potent and selectiveSEW2871/Tocris 2 μM at S6-S7 3-[3-(trifluoromethyl)phenyl]1,2,4-sphingosine-1- Bioscience/2284 oxadiazole/256414-75-2/C₂₀H₁₀F₆N₂OSphosphate 1 (S1P1) receptor agonist[9S-(9α,10β,11β,13α)]-2,3,10,11,12,13- Broad spectrum proteinStaurosporine/Tocris 10 nM at S6-S7 Hexahydro-10-methoxy-9-methyl-11-kinase inhibitor Bioscience/1285 (methylamino)-9,13-epoxy-1H,9H-diindolo[1,2,3-gh:3′,2′,1′-lm]pyrrolo[3,4-j][1,7]benzodiazonin-1-one/62996-74- 1/C₂₀H₂₆N₄O₃

As shown in FIGS. 8A, 8B, 8C, 8D and 8E, which are graphs depicting datafrom real-time PCR analyses of the expression of insulin and MAFA aftertreatment with small molecules, addition of EPZ-5676 (inhibitor ofprotein methyltransferase DOT1L) and AXL inhibitor (R428) significantlyupregulated expression of MAFA as compared to untreated cultures atStage 6 and Stage 7, respectively.

Example 3 Prophetic Generation of Endocrine Cells Co-Expressing Insulin,PDX-1, NKX6.1, and MAFA in Suspension Cultures

Cells of the human embryonic stem cell line H1 (WA01) are seeded assingle cells at 1×10⁵ cells/cm² on MATRIGEL™ (1:30 dilution)-coateddishes in a media comprising of DMEM-F12, GlutaMAX™ (1:100 dilution),0.25 mM ascorbic acid, 100 ng/ml of FGF2 (R & D systems, MN), 1 ng/ml ofTGF-β, ITS-X (1:100 dilution), 2% FAF-BSA, and 20 ng/ml of IGF-1,supplemented with 10 μM of Y-27632. Forty-eight hours post-seeding, thecultures are washed in incomplete PBS (phosphate buffered saline withoutMg or Ca) followed by incubation with 1× TrypLE™ Express Enzyme for 3 to5 minutes at 37° C. The released cells are rinsed with DMEM-F12 and spunat 1000 rpm for 5 minutes. The resulting cell pellet are resuspended inDMEM-F12 supplemented with 10 μM Y-27632, 1× GlutaMAX™, 0.25 mM ascorbicacid, 100 ng/ml FGF2, 1 ng/ml TGF-β, ITS-X at a (1:100 dilution, 2%FAF-BSA, and 20 ng/ml of IGF-1 and the single cell suspension seeded atapproximately 1.3 to 1.5×10⁵ cells/cm². The cultures are fed every daywith medium and differentiation, according to the following protocol,was initiated 48 hrs. following seeding resulting in an about 90%starting confluency. Stage 1 through Stage 4 are maintained on planaradherent cultures while Stages 5 through 7 are maintained in suspensioncultures.

Stage 1 (3 Days):

Cells are plated on MATRIGEL™ (1:30 dilution)-coated dishes were firstrinsed with 1× incomplete DPBS and then are cultured for one day inStage 1 media: MCDB-131 medium supplemented with 0.5% FAF-BSA, 1.2g/1000 ml sodium bicarbonate; 1× concentration of GlutaMAX™; 4.5 mMD-glucose; 100 ng/ml GDF8; and 1 μM MCX compound. Cells are thencultured for an additional day in MCDB-131 medium supplemented with 0.5%FAF-BSA, 1.2 g/1000 ml sodium bicarbonate, 1× concentration ofGlutaMAX™, 4.5 mM D-glucose, 100 ng/ml GDF8, and 0.1 μM MCX compound.Cells are then cultured for an additional day in MCDB-131 mediumsupplemented with 0.5% FAF-BSA, 1.2 g/1000 ml sodium bicarbonate, 1×concentration of GlutaMAX™, 4.5 mM D-glucose, and 100 ng/ml GDF8.

Stage 2 (2 Days):

Cells are first rinsed with 1× incomplete DPBS and then are treated fortwo days with MCDB-131 medium supplemented with 0.5% FAF-BSA; 1.2 g/1000ml sodium bicarbonate; 1× concentration of GlutaMAX™; 4.5 mM D-glucose;0.25 mM ascorbic acid and 25 ng/ml FGF7.

Stage 3 (2 Days):

Cells are treated with BLAR custom medium supplemented with a 1:200dilution of ITS-X; 4.5 mM glucose; 1× concentration of GlutaMAX™; 2.5g/1000 ml sodium bicarbonate; 2% FAF-BSA; 0.25 μM SANT-1; 1 μM RA; 25ng/ml FGF7; 0.25 mM ascorbic acid; 200 nM TPB; and 100 nM LDN-193189 fortwo days.

Stage 4 (3 Days):

Cells are treated with BLAR medium supplemented with a 1:200 dilution ofITS-X; 4.5 mM glucose; 1× concentration of GlutaMAX™; 2.5 g/1000 mlsodium bicarbonate; 2% FAF-BSA; 0.25 μM SANT-1; 100 nM RA; 2 ng/ml FGF7;100 nM LDN-193189; 0.25 mM ascorbic acid; and 200 nM TPB for three days,then at end of Stage 4 cells cultured on planar dishes are treated for 4hours with 10 μM of Y-27632, are rinsed with PBS and are treated for 5minutes at room temperature with 1× concentration of StemPro® Accutase®enzyme (Life Technologies, #A11105-01) and the enzyme is removed, andare rinsed with BLAR basal media and cells are scraped using a cellscraper and broken into cell clusters (<100 micron). The cell clustersare transferred into a disposable polystyrene 125 ml Spinner Flask(Corning), and spun at 80 to 100 rpm in suspension with Stage 5 mediaspecified below.

Stage 5 (3 Days):

Stage 4 cells are prepared as clusters are cultured in suspension inBLAR medium supplemented with a 1:200 dilution of ITS-X; 20 mM glucose(final); 1× GlutaMAX™; 1.5 g/1000 ml sodium bicarbonate; 2% FAF-BSA;0.25 mM ascorbic acid; 10 μg/ml of heparin, 10 μM ZnSO₄, 0.25 μM SANT-1;50 nM RA; 100 nM LDN-193189; 1 μM of T3 as 3,3′,5-triiodo-L-thyroninesodium salt, and 10000 nM of ALK5 inhibitor II for three days.

Stage 6 (7 Days):

Stage 5 cells are cultured in suspension and treated with BLAR mediumsupplemented with a 1:200 dilution of ITS-X; 20 mM Glucose (final); 1×concentration of GlutaMAX™; 1.5 g/1000 ml sodium bicarbonate; 2%FAF-BSA; 0.25 mM ascorbic acid; 10 μg/ml of heparin, 10 μM ZnSO₄, 100 nMLDN-193189, 1 μM T3 as 3,3′,5-triiodo-L-thyronine sodium salt, 100 nMgamma secretase inhibitor XX, and 10000 nM ALK5 inhibitor II for 7 days.

Stage 7 (15 Days):

Stage 6 cells are cultured in suspension and are treated with BLARmedium supplemented with a 1:200 dilution of ITS-X; 20 mM Glucose(final); 1× concentration of GlutaMAX™; 0.0015 g/ml sodium bicarbonate;2% FAF-BSA; 10 μg/ml of heparin, 10 μM ZnSO₄, 1 μM T3 as3,3′,5-triiodo-L-thyronine sodium salt, 10000 nM ALK5 inhibitor II, 10μM Trolox, 1 mM N-acetyl cysteine, and 2 μM AXL inhibitor (R428) for upto 15 days.

At Stages 5-7, aliquots of cell clusters are removed and characterizedby PCR, FACS and immune histochemistry for co-expression of insulin,NKX6.1, PDX-1, and MAFA. It is expected that the results of such testingwill show co-expression of insulin, PDX1, NKX6.1 and MAFA within thesame cell and a population of cells in which at least about 10% of thecell population showed such expression.

Example 4 mRNA Expression of AXL and Co-Ligand GAS6 is Very Low forStage 7 or Human Islet Cells

Cells of the human embryonic stem cell line H1 (WA01) were seeded assingle cells at 1×10⁵ cells/cm² on MATRIGEL™ (1:30 dilution)-coateddishes in a media comprising of Essential 8™ (“E8”) (BD Biosciences;Catalog No. 356231). At 48 hours post-seeding, the cultures were washedin 1× incomplete PBS followed by incubation with 1× TrypLE™ ExpressEnzyme (Life Science; Catalog No. 14190) for 3 to 5 minutes at 37° C.The released cells were rinsed with E8 and spun at 1000 rpm for 5minutes. The resulting cell pellet was resuspended in E8 supplementedwith 10 μM Y-27632 and the single cell suspension was seeded atapproximately 1.3 to 1.5×10⁵ cells/cm². The cultures were fed every daywith E8 medium and differentiation, according to the following protocol,was initiated 48 hrs. following seeding resulting in an about 90%starting confluency.

Stage 1 (3 Days):

Cells were plated on MATRIGEL™ (1:30 dilution)-coated dishes were firstrinsed with 1× incomplete DPBS and then cultured for one day in Stage 1media: MCDB-131 medium supplemented with 0.5% FAF-BSA, 1.5 g/1000 mlsodium bicarbonate; 1× concentration of Glutamax™, 4.5 mM D-glucose; 100ng/ml GDF8; and 1.5 μM MCX compound. Cells were then cultured for anadditional day in MCDB-131 medium supplemented with 0.5% FAF-BSA, 1.5g/1000 ml sodium bicarbonate, 1× concentration Glutamax™, 4.5 mMD-glucose concentration, 100 ng/ml GDF8, and 0.1 μM MCX compound. Cellswere then cultured for an additional day in MCDB-131 medium supplementedwith 0.5% FAF-BSA, 1.5 g/1000 ml sodium bicarbonate, 1× concentration ofGlutamax™, 4.5 mM D-glucose, and 100 ng/ml GDF8.

Stage 2 (2 Days):

Cells were first rinsed with 1× incomplete DPBS and then cultured fortwo days with MCDB-131 medium supplemented with 0.5% FAF-BSA; 1.5 g/1000ml sodium bicarbonate; 1× concentration of Glutamax™, 4.5 mM D-glucose;0.25 mM ascorbic acid and 50 ng/ml FGF7.

Stage 3 (2 Days):

Cells were cultured in BLAR custom medium supplemented with a 1:100dilution of ITS-X; 1× concentration Glutamax™, 4.5 mM D-glucose; 2.5g/1000 ml sodium bicarbonate; 2% FAF-BSA; 0.25 μM SANT-1; 1 μM RA; 25ng/ml FGF7; 0.25 mM ascorbic acid; 300 nM TPB; and 100 nM LDN-193189 fortwo days.

Stage 4 (3 Days):

Cells were cultured in BLAR medium supplemented with a 1:100 dilution ofITS-X; 1× concentration Glutamax™, 4.5 mM D-glucose; 2.5 g/1000 mlsodium bicarbonate; 2% FAF-BSA; 0.25 μM SANT-1; 0.1 μM RA; 2 ng/ml FGF7;100 nM LDN-193189; 0.25 mM ascorbic acid; and 200 nM TPB for three days,then at end of Stage 4 cells cultured on planar dishes were treated for4 hours with 10 μM of Y-27632, rinsed with 1× incomplete PBS and treatedfor 3 to 5 minutes at room temperature with 1× TrypLE™. The enzyme wasremoved, the cells released and rinsed with BLAR media and transferredinto a disposable polystyrene 125 ml Spinner Flask, and spun at 1000 rpmfor 3 mins. The resulting cell pellet was resuspended as single cells ata density of approximately 0.5×10⁵ cells/cm² on filter inserts (BDBiosciences; Catalog No. 3420) (5 to 10 μL per spot for a total of 0.25to 0.5 million cells/spot). Each spotted area measured approximately 1to 2 mm in diameter depending on the volume of cells added. For 6-wellinserts, 1.5 mL/well was added to the bottom of each insert whereas 8 mLwas added for 10 cm filter inserts. Typically, 20 to 15 spots were usedper well of a 6-well insert and 80 to 90 spots were used for 10 cminserts.

Stage 5 (3 Days):

Stage 4 cells were cultured in BLAR medium supplemented with a 1:100dilution of ITS-X; 20 mM glucose (final); 1.5 g/1000 ml sodiumbicarbonate; 2% FAF-BSA; 10 μg/ml of heparin, 10 μM ZnSO₄, 0.25 μMSANT-1; 0.05 μM RA; 100 nM LDN-193189, 1 μM of T3 as3,3′,5-triiodo-L-thyronine sodium salt, and 10 μM of ALK5 inhibitor IIfor three days.

Stage 6 (7 Days):

Stage 5 cells were cultured in BLAR medium supplemented with a 1:100dilution of ITS-X; 20 mM glucose (final); 1.5 g/1000 ml sodiumbicarbonate; 2% FAF-BSA; 10 μg/ml of heparin, 10 μM ZnSO₄, 100 nMLDN-193189, 1 μM T3 as 3,3′,5-triiodo-L-thyronine sodium salt, 100 nMgamma secretase inhibitor XX, and 10 μM ALK5 inhibitor II for 7 days.

Stage 7 (7 days):

Stage 6 cells were cultured in BLAR medium supplemented with a 1:100dilution of ITS-X; 20 mM glucose (final); 1.5 g/L sodium bicarbonate; 2%FAF-BSA; 10 μg/ml of heparin, 10 μM ZnSO₄, 1 μM T3 as3,3′,5-triiodo-L-thyronine sodium salt, 10 μM ALK5 inhibitor II, 1 mMN-acetyl cysteine, and 2 μM AXL inhibitor (R428) for 7 days.

At Stage 4, day 3, Stage 5, day 3, Stage 6, day 3, and Stage 7, day 7mRNA was collected and expression of AXL and GAS 6 evaluated as comparedto undifferentiated human stem cells and cadaveric human islets (ProdoLabs, California). As depicted in FIG. 9, expression of AXL was presentat a high level in undifferentiated stem cells. However, differentiationof stem cells towards pancreatic endoderm, pancreatic endocrine andimmature beta cells resulted in a precipitous drop in AXL expression.Moreover, there was a low level of GAS6 expression maintained at Stages4 through 7. Expression of AXL was also significantly lower in humanislets as compared to undifferentiated stem cells. The results show thatStage 6 and 7 cells have very low expression of AXL.

Example 5 R428 Inhibits AXL and Many Additional Kinases

The efficiency of the AXL inhibitor R428 for targeting different kinaseswas assessed by Kinase Profiling Services using 100 μM ATP concentration(EMD Millipore). R428 was tested at 1 and 10 μM. Table IV lists thekinases profiled along with the efficiency in targeting the kinases witha lower number indicating a more robust inhibition of a particularkinase.

TABLE IV Kinase profiling of R428 R428 R428 Kinase 1001010 @ 1 μM1001010 @ 10 μM ALK4(h) 84 48 Aurora-A(h) 20 3 Aurora-B(h) 1 0 Axl(h) −1−1 Blk(h) 27 2 CaMKIIβ(h) 76 5 CaMKIδ(h) 76 24 CDK1/cyclinB(Hh) 101 88CDK5/p35(h) 101 94 CHK1(h) 71 23 CHK2(h) 47 9 CK2(h) 103 106 CK2α2(h)112 97 CLK2(h) 78 28 cSRC(h) 51 10 EGFR(h) 89 40 Eph A 2(h) 54 12FGFR1(h) 17 1 Flt3(h) 9 1 GSK3α(h) 95 105 GSK3β(h) 106 99 IGF-1R(h) 9358 IKKβ(h) 86 53 IR(h) 83 24 IRAK4(h) 94 51 JAK2(h) 96 41 JAK3(h) 69 18MAPK1(h) 108 106 Met(h) 49 −1 NEK2(h) 60 10 PAK4(h) 97 73 PDGFRβ(h) 3721 Pim-2(h) 87 74 PKA(h) 88 35 PKBα(h) 87 57 PKCα(h) 101 96 PKCβ1(h) 10295 Plk1(h) 83 65 Plk3(h) 101 81 Ret(h) 1 1 ROCK-1(h) 93 41 Rsk3(h) 2 1SAPK3(h) 106 108 SAPK4(h) 94 91 TGFBR1(h) 97 69 TrkC(h) 47 13 ZAP-70(h)92 66 ZIPK(h) 97 46

The kinase profiling results indicate that R428 inhibits AXL asexpected. However, additionally R428 potently inhibits RSK3, Ret, Flt,FGFrl, AuroraA and AuroraB kinases at 1 and 10 μM. This signifies thatthe mechanism of R428 action in induction of MAFA, may not be throughAXL receptor inhibition. In fact, the examples herein show that mRNAexpression for AXL at Stage 7 is very low highlighting that themechanism of action of R428 in inducing MAFA expression is not throughinhibition of AXL, but rather through the inhibition of other kinases,such as RSK3 and aurora kinases.

Example 6 Inhibition of Aurora Kinase Expression Enhanced MAFAExpression at Stage 7 in the Absence of R428

Cells of the human embryonic stem cell line H1 (WA01) were seeded assingle cells at 1×10⁵ cells/cm² on MATRIGEL™ (1:30 dilution) coateddishes in E8 media. At about 70 to 80% confluency, the cultures werewashed in 1× incomplete DPBS followed by incubation with 1×TrypLE™Express Enzyme for 3 to 5 minutes at 37° C. The released cells wererinsed with E8 and spun at 1000 rpm for 5 minutes. The resulting cellpellet was resuspended in E8 supplemented with 10 μM Y-27632 and thesingle cell suspension was seeded at approximately 1.3 to 1.5×10⁵cells/cm². The cultures were fed every day with E8 medium anddifferentiation, according to the following protocol, was initiated 48hrs. following seeding resulting in an about 90% starting confluency.

Stage 1 (3 Days):

Cells were plated on MATRIGEL™ (1:30 dilution) coated dishes were firstrinsed with 1× incomplete DPBS and then cultured for one day in Stage 1media: MCDB-131 medium supplemented with 0.5% FAF-BSA, 1.5 g/1000 mlsodium bicarbonate; 10 mM final glucose concentration; 100 ng/ml GDF8;and 1.5 μM MCX compound. Cells were then cultured for an additional dayin MCDB-131 medium supplemented with 0.5% FAF-BSA, 1.5 g/1000 ml sodiumbicarbonate, 10 mM final glucose concentration, 100 ng/ml GDF8, and 0.1μM MCX compound. Cells were then cultured for an additional day inMCDB-131 medium supplemented with 0.5% FAF-BSA, 1.5 g/1000 ml sodiumbicarbonate, 10 mM final glucose concentration, and 100 ng/ml GDF8.

Stage 2 (2 Days):

Cells were rinsed with 1× incomplete DPBS and then cultured for two dayswith MCDB-131 medium supplemented with 0.5% FAF-BSA; 1.5 g/1000 mlsodium bicarbonate; 10 mM final glucose concentration; 0.25 mM ascorbicacid and 50 ng/ml FGF7.

Stage 3 (2 Days):

Cells were cultured in BLAR custom medium supplemented with a 1:100dilution of ITS-X; 10 mM final glucose concentration; 2.5 g/1000 mlsodium bicarbonate; 2% FAF-BSA; 0.25 μM SANT-1; 1 μM RA; 25 ng/ml FGF7;0.25 mM ascorbic acid; 300 nM TPB; and 100 nM LDN-193189 for two days.

Stage 4 (3 Days):

Cells were cultured in BLAR medium supplemented with a 1:100 dilution ofITS-X; 10 mM final glucose concentration; 2.5 g/1000 ml sodiumbicarbonate; 2% FAF-BSA; 0.25 μM SANT-1; 0.1 μM RA; 2 ng/ml FGF7; 100 nMLDN-193189; 0.25 mM ascorbic acid; and 200 nM TPB for three days, thenat end of Stage 4 cells cultured on planar dishes were treated for 4hours with 10 μM of Y-27632, rinsed with 1× incomplete PBS and treatedfor 3 to 5 minutes at room temperature with 1× TrypLE™. The enzyme wasremoved, the cells released and rinsed with BLAR media and transferredinto a disposable polystyrene 125 ml Spinner Flask, and spun at 1000 rpmfor 3 mins. The resulting cell pellet was resuspended as single cells ata density of approximately 0.5×10⁵ cells/cm² on filter inserts (5 to 10μl per spot for a total of 0.25 to 0.5 million cells/spot). Each spottedarea measured approximately 1 to 2 mm in diameter depending on thevolume of cells added. For 6-well inserts, 1.5 mL/well was added to thebottom of each insert whereas 8 mL was added for 10 cm filter inserts.Typically, 20 to 15 spots were used per well of a 6-well insert ad 80 to90 spots were used for 10 cm inserts.

Stage 5 (3 Days):

Stage 4 cells were cultured in BLAR medium supplemented with a 1:100dilution of ITS-X; 20 mM glucose (final); 1.5 g/1000 ml sodiumbicarbonate; 2% FAF-BSA; 10 μg/ml of heparin, 10 μM ZnSO₄, 0.25 μMSANT-1; 0.05 μM RA; 100 nM LDN-193189, 1 μM of T3 as3,3′,5-triiodo-L-thyronine sodium salt, and 10 μM of ALK5 inhibitor IIfor three days.

Stage 6 (7 Days):

Stage 5 cells were cultured in BLAR medium supplemented with a 1:100dilution of ITS-X; 20 mM glucose (final); 1.5 g/1000 ml sodiumbicarbonate; 2% FAF-BSA; 10 μg/ml of heparin, 10 μM ZnSO₄, 100 nMLDN-193189, 1 μM T3 as 3,3′,5-triiodo-L-thyronine sodium salt, 100 nMgamma secretase inhibitor XX, and 10 μM ALK5 inhibitor II for 7 days.

Stage 7 (7 Days):

Stage 6 cells were cultured for seven days in BLAR medium supplementedwith a 1:100 dilution of ITS-X; 20 mM glucose (final); 1.5 g/L sodiumbicarbonate; 2% FAF-BSA; 10 μg/ml of heparin, 10 μM ZnSO₄, 1 μM T3 as3,3′,5-triiodo-L-thyronine sodium salt, 10 μM ALK5 inhibitor II, 1 mMN-acetyl cysteine. Some cultures also included one of 2 μM R428, 2 μMaurora kinase inhibitor VI(4-(4-(N-Benzoylamino)anilino)-6-methoxy-7-(3-(1-morpholino)propoxy)quinazoline)(EMD Millipore; Catalog No, 18941), or 2 μM aurora kinase inhibitor II(4-(4′-Benzamidoanilino)-6,7-dimethoxyquinazoline) (EMD Millipore;Catalog No. 189404).

At Stage 7, day 7, mRNA was collected and expression of MAFA, UCN3,PDX1, NKX6.1, insulin and G6PC2 evaluated as compared toundifferentiated human stem cells. As depicted in FIGS. 10A, 10B, 10C,10D, 10E and 10F, removal of R428 resulted in a significant decrease inMAFA expression. A significant rise in UCN3 and G6PC2 expression, bothmarkers of mature beta cells, was noted for cultures not treated withR428 suggesting that, although R428 increases MAFA expression, thecompound reduces other beta cell maturation markers. Substitution ofaurora kinase inhibitors for R428 restored MAFA expression while notdecreasing G6PC2 levels. Thus, the induction of MAFA expression by R428at Stage 7 was likely not through AXL inhibition, but rather throughinhibition of aurora kinases. The use of aurora kinase inhibitor IIresulted in an increase in MAFA expression and maintenance of UCN3 andG6PC2 expression.

Example 7 Inhibition of Aurora Kinase or RSK Enhanced Expression of MAFAExpression at Stage 7 in the Absence of R428

Cells of the human embryonic stem cell line H1 (WA01) were seeded assingle cells at 1×10⁵ cells/cm² on MATRIGEL™ (1:30 dilution) coateddishes in E8 media. At about 70 to 80% confluency, the cultures werewashed in 1× incomplete DPBS followed by incubation with 1× TrypLE™Express Enzyme for 3 to 5 minutes at 37° C. The released cells wererinsed with E8 and spun at 1000 rpm for 5 minutes. The resulting cellpellet was resuspended in E8 supplemented with 10 μM Y-27632 and thesingle cell suspension was seeded at approximately 1.3 to 1.5×10⁵cells/cm². The cultures were fed every day with E8 medium anddifferentiation, according to the following protocol, was initiated 48hrs. following seeding resulting in an about 90% starting confluency.

Stage 1 (3 Days):

Cells were plated on MATRIGEL™ (1:30 dilution) coated dishes were firstrinsed with 1× incomplete DPBS and then cultured for one day in Stage 1media: MCDB-131 medium supplemented with 0.5% FAF-BSA, 1.5 g/1000 mlsodium bicarbonate; 10 mM final glucose concentration; 100 ng/ml GDF8;and 1.5 μM MCX compound. Cells were then cultured for an additional dayin MCDB-131 medium supplemented with 0.5% FAF-BSA, 1.5 g/1000 ml sodiumbicarbonate, 10 mM final glucose concentration, 100 ng/ml GDF8, and 0.1μM MCX compound. Cells were then cultured for an additional day inMCDB-131 medium supplemented with 0.5% FAF-BSA, 1.5 g/1000 ml sodiumbicarbonate, 10 mM final glucose concentration, and 100 ng/ml GDF8.

Stage 2 (2 Days):

Cells were rinsed with 1× incomplete DPBS and then cultured for two dayswith MCDB-131 medium supplemented with 0.5% FAF-BSA; 1.5 g/1000 mlsodium bicarbonate; 10 mM final glucose concentration; 0.25 mM ascorbicacid and 50 ng/ml FGF7.

Stage 3 (2 Days):

Cells were cultured in BLAR custom medium supplemented with a 1:100dilution of ITS-X; 10 mM final glucose concentration; 2.5 g/l 000 mlsodium bicarbonate; 2% FAF-BSA; 0.25 μM SANT-1; 1 μM RA; 25 ng/ml FGF7;0.25 mM ascorbic acid; 300 nM TPB; and 100 nM LDN-193189 for two days.

Stage 4 (3 Days):

Cells were cultured in BLAR medium supplemented with a 1:100 dilution ofITS-X; 10 mM final glucose concentration; 2.5 g/1000 ml sodiumbicarbonate; 2% FAF-BSA; 0.25 μM SANT-1; 0.1 μM RA; 2 ng/ml FGF7; 100 nMLDN-193189; 0.25 mM ascorbic acid; and 200 nM TPB for three days, thenat end of Stage 4 cells cultured on planar dishes were treated for 4hours with 10 μM of Y-27632, rinsed with 1× incomplete PBS and treatedfor 3 to 5 minutes at room temperature with 1× TrypLE™. The enzyme wasremoved, the cells released and rinsed with BLAR media and transferredinto a disposable polystyrene 125 ml Spinner Flask, and spun at 1000 rpmfor 3 mins. The resulting cell pellet was resuspended as single cells ata density of approximately 0.5×10⁵ cells/cm² on filter inserts (5 to 10μl per spot for a total of 0.25 to 0.5 million cells/spot). Each spottedarea measured approximately 1 to 2 mm in diameter depending on thevolume of cells added. For 6-well inserts, 1.5 mL/well was added to thebottom of each insert whereas 8 mL was added for 10 cm filter inserts.Typically, 20 to 15 spots were used per well of a 6-well insert ad 80 to90 spots were used for 10 cm inserts.

Stage 5 (3 Days):

Stage 4 cells were cultured in BLAR medium supplemented with a 1:100dilution of ITS-X; 20 mM glucose (final); 1.5 g/1000 ml sodiumbicarbonate; 2% FAF-BSA; 10 μg/ml of heparin, 10 μM ZnSO₄, 0.25 μMSANT-1; 0.05 μM RA; 100 nM LDN-193189, 1 μM of T3 as3,3′,5-triiodo-L-thyronine sodium salt, and 10 μM of ALK5 inhibitor IIfor three days.

Stage 6 (7 Days):

Stage 5 cells were cultured in BLAR medium supplemented with a 1:100dilution of ITS-X; 20 mM glucose (final); 1.5 g/1000 ml sodiumbicarbonate; 2% FAF-BSA; 10 μg/ml of heparin, 10 μM ZnSO₄, 100 nMLDN-193189, 1 μM T3 as 3,3′,5-triiodo-L-thyronine sodium salt, 100 nMgamma secretase inhibitor XX, and 10 μM ALK5 inhibitor II for 7 days.

Stage 7 (7 Days):

Stage 6 cells were cultured for fourteen days in BLAR mediumsupplemented with a 1:100 dilution of ITS-X; 20 mM glucose (final); 1.5g/L sodium bicarbonate; 2% FAF-BSA; 10 μg/ml of heparin, 10 M ZnSO₄, 1μM T3 as 3,3′,5-triiodo-L-thyronine sodium salt, 10 μM ALK5 inhibitorII, 1 mM N-acetyl cysteine. Some cultures also included one of 2 μMR428, 2-5 μM RSK inhibitor II(2-(3,5-Difluoro-4-hydroxy-anilino)-8-isopentyl-5,7-dimethyl-7H-pteridin-6-one)(EMD Millipore; Catalog No, 559286-5MG), 2-5 μM aurora kinase inhibitorII (EMD Millipore, or a combination of 2-5 μM RSK inhibitor II and 2-5μM aurora kinase II inhibitor.

At Stage 7, day 14, mRNA was collected and compared to undifferentiatedhuman stem cells. As depicted in FIG. 1, removal of R428 resulted in asignificant decrease in MAFA expression. Substitution with aurora kinaseinhibitor II for R428 restored MAFA expression. Similarly, substitutionwith RSK inhibitor for R428 restored MAFA expression. Substitution withaurora kinase inhibitor II and RSK inhibitor for R428 further enhancedMAFA expression. This data indicates that the induction of MAFAexpression by R428 was likely not through AXL inhibition, but ratherthrough inhibition of aurora kinase, RSK or a combination thereof.

What is claimed is:
 1. An in vitro cell culture, comprising a populationof differentiated pluripotent stem cells expressing markerscharacteristics of pancreatic endocrine cells, wherein at least 10% ofthe differentiated cells express insulin, PDX1 NKX6.1, and MAFA.
 2. Amethod of inducing MAFA expression in cells derived from pluripotentcells, comprising culturing pluripotent cells, differentiating thepluripotent cells into pancreatic endocrine cells of a more maturephenotype by treatment with a medium supplemented with an inhibitorselected from the group consisting of an aurora kinase inhibitor, an RSKinhibitor, An inhibitor of protein methyltransferase DOT1L, andcombinations thereof.
 3. The method of claim 2, wherein the inhibitor isan aurora kinase inhibitor.
 4. The method of claim 3, wherein aurorakinase inhibitor is aurora kinase inhibitor II.
 5. The method of claim2, wherein the inhibitor is an RSK inhibitor.
 6. The method of claim 3,wherein aurora kinase inhibitor is RSK inhibitor II.
 7. The method ofclaim 2, wherein the medium further comprises an antioxidant.